JP7089474B2 - Immunological reagents - Google Patents

Description

Cross-reference of related applications

This application is incorporated herein by reference in its entirety, US Patent Application No. 62 / 286,269, filed January 22, 2016, and US, filed February 3, 2016. Claim the priority benefit of Patent Application No. 62 / 290,745.

The present disclosure discloses a binding agent having specificity for Programmed Cell Death 1 (PD-1) (eg, human PD-1), as well as infectious diseases (eg, human immunodeficiency virus (HIV)), cancer and / or autoimmune. It relates to a method of using it to treat and / or prevent immunity.

In the 40th year since the HIV epidemic began, the understanding of the etiology of HIV has progressed, and the development of powerful and safe antiviral drugs has made great progress. With more than 30 antiviral drugs registered, the impact of combination antiretroviral therapy (ART) on morbidity and mortality is significant. However, despite long-term suppression of HIV replication has been achieved in patients who have optimally adhered to ART, HIV always reverts after discontinuation of treatment. Moreover, successful therapies do not induce, or allow recovery / development of, virus-specific immune responses that can control HIV replication in the absence of ART. Therefore, lifelong ART is required to control HIV replication and related diseases in most HIV-infected subjects.

A long-lived central memory CD4 T cell population latently infected with HIV has been identified in the blood as an important component of the HIV cell reservoir and as a major cause of HIV persistence. The lifespan of this latent cell reservoir is estimated to be approximately 70 years in the presence of complete HIV suppression using ART. However, recent studies have demonstrated that two populations of CD4 T cells residing in the lymph nodes function as the major CD4 T cell compartments for HIV infection, replication and production. These two CD4 T cell populations are defined by expression of PD-1 and CXCR5, PD-1 ⁺ CXCR5 ⁺ , that is, follicular helper T cells (Tfh), and PD-1 ⁺ CXCR5 ^- CD4 T cell populations. include.

Numerous mechanisms have been proposed that are responsible for the establishment and maintenance of HIV latent cell reservoirs. One of the mechanisms is the persistence of minimal viral replication under ART, which can replace the HIV cell reservoir. Therefore, ART cannot induce complete suppression of HIV replication, nor can the "natural" HIV-1-specific immune response under ART completely suppress and eliminate ongoing residual viral replication. .. Failure of ART and HIV-specific immune responses provides a rationale for considering alternative interventions that also target permanent HIV cell reservoirs.

Numerous immunological interventions have been considered to date, but are still under development with the goal of achieving functional cure of HIV, which suppresses viral replication without sustained antiviral therapy. (9). Therapeutic vaccine strategies were the primary intervention strategy considered, but the results were in laboratory animal models and patients, with the exception of the CMV-based vector HIV vaccine (50% efficacy in the NHP model; 10). It shows only a slight effectiveness. Recent studies have yielded interesting results regarding the potential use of anti-enveloped widespread neutralizing antibodies (bNabs) as therapeutic agents in HIV infection (11, 12). In addition, antagonist PD-1 antibody (Ab) has been shown to restore T cell function in HIV-infected patients and uses these antibodies as a therapeutic strategy to enhance the efficacy of HIV-specific T cell responses. Possibility has been proposed (13, 14).

It is well documented that invasive tumor-specific CD8 T cells are dysfunctional in terms of their ability to proliferate and mediate cytotoxic activity. Most invasive tumor-specific CD8 T cells are in a so-called functionally exhausted state. The major mechanism responsible for the exhaustion of invasive tumor-specific CD8 T cells is increased expression of numerous regulatory receptors and in particular PD-1 regulatory receptors. The finding that blocking of PD-1 / PDL-1 / 2 (PD-1 ligand) is associated with recovery from exhaustion of CD8 T cells is that PD-1 expressed by exhausted CD8 T cells. It provides a rationale for developing intervention strategies targeting molecules. Recent studies have shown highly promising results with the use of PD-1 antibody with antagonist activity in patients with advanced cancer-related diseases. Studies have shown a substantial response rate in the range of 18-40% in patients with advanced melanoma, non-small cell lung cancer and renal cancer. In these tests, anti-PD-1 antibody was used alone or in combination with anti-CTL-A4 antibody. Following these initial trials, current trials are being conducted in patients with a variety of tumors, including hematological malignancies.

There is a demand in the art for additional reagents and methods of using them for targeting PD-1. The present disclosure addresses those requirements by providing reagents and methods that can be used to target PD-1 and the cells and / or tissues expressing it.

The present disclosure discloses a binding agent having specificity for Programmed Cell Death 1 (PD-1) (eg, human PD-1), as well as, for example, an infectious disease (eg, due to a human immunodeficiency virus (HIV)). It relates to methods of using it to treat, prevent and / or ameliorate cancer and / or autoimmune and / or neurodegenerative conditions. Functional assays are also provided to identify binders that interact with PD-1. Combinations of binders are also provided, such as a first binder that blocks the interaction between PD-1 and PD-L1 and a second binder that does not block the interaction between PD-1 and PD-L1. They act synergistically to relieve T cells from exhaustion.

Antibody concentration reaction curves (A to C) for inhibition of PD-1 / PD-L1 interaction. Structural representation of the ectodomain of PD-1 protein from residues 33-149. The amino acids involved in the interaction with either PD-L1 or PD-L2 are at structural positions indicated by the purple circle along with the residue number (residue number corresponds to SEQ ID NO: 275 (FIG. 3H)). .. It is an amino acid sequence of PD-1 ectodomain. Residues targeted for amino acid substitutions are shown for each of the 31 mutations (M1-M31). The residues described in purple letters correspond to amino acids involved in PD-1 / PD-L1 interaction, and the asparagine residues shown in green letters are positions that are candidates for N-linked glycosylation. It is a modified PD-1 polypeptide. It is a modified PD-1 polypeptide. It is a modified PD-1 polypeptide. It is a modified PD-1 polypeptide. It is a modified PD-1 polypeptide. It is a modified PD-1 polypeptide. It is an amino acid sequence of human PD-1 (SEQ ID NO: 275). It is an amino acid sequence of monkey PD-1. Blocking and non-blocking anti-PD-1 antibodies against modified PD-1 proteins M13, M14 and M23 expressed on the surface of transiently transfected HeLa cells, as well as wild-type (WT) controls (SEQ ID NO: 275). Combine. Blocking and non-blocking anti-PD-1 antibodies against modified PD-1 proteins M13, M23 and M4 expressed on the surface of transiently transfected HeLa cells, as well as wild-type (WT) controls (SEQ ID NO: 275). Combine. Blocking and non-blocking anti-PD-1 antibodies against modified PD-1 proteins M31, M5 and M18 expressed on the surface of transiently transfected HeLa cells, as well as wild-type (WT) controls (SEQ ID NO: 275). Combine. Blocking against modified PD-1 proteins M1, M9, M13, M17, M18, M23, and M28 expressed on the surface of transiently transfected HeLa cells, as well as wild-type (WT) controls (SEQ ID NO: 275). And binding of non-blocking anti-PD-1 antibodies. Blocking and non-blocking anti-PD-1 antibodies against modified PD-1 proteins M17, M30 and M26 expressed on the surface of transiently transfected HeLa cells, as well as wild-type (WT) controls (SEQ ID NO: 275). Combine. The combination of two antagonist anti-PD-1 antibodies that bind to different epitopes on PD-1 (one blocking and the other non-blocking to PD-1 / PD-L1 interactions) is either antibody alone. To the extent achieved in, enhances the relief of functional exhaustion of HIV-specific CD8 T cells and enhances their proliferation. Individual antagonist anti-PD-1 antibodies, as well as a combination of two antagonist anti-PD-1 antibodies that bind to different epitopes on PD-1, proliferate PD-1 ⁺ memory CD4 T cells in a mixed lymphocyte reaction assay. a) and enhance IFNγ production (b). Structural representation of the evolutionarily preserved patches P1 (a) and P2 (b) of PD-1. Alignment of amino acid sequences of PD-1 ectodomains from different species (c). Antibody competitive binding test against cell surface PD-1 on activated CD4 ⁺ T cells. Epitope mapping of antibody binding sites to PD-1 structure using a combination of both site-specific mutagenesis and antibody competitive binding results (a and b). Cell death in PD-1 high CD4 T cells from viralemia HIV-infected donors when treated with anti-PD-1 ADC compared to anti-PD-1 antibody alone or IgG control antibody (Aqua staining (a)). Or selective increase in apoptosis (Annexin V staining (b)). Anti-PD-1 antibody drug conjugate (ADC) treatment involves apoptosis (Annexin V staining (a)) and / or cell death (Aqua) in PD-1 high CD4 ⁺ T cells from multiple different viremia HIV-infected donors. It results in an increase in staining (b)). Low cell death / apoptosis is observed in control samples treated with either anti-PD-1 antibody alone or IgG1 control antibody. Assessment of PD-1 positive infected CD4 ⁺ T cell anti-PD-1 antibody drug conjugate (ADC) -mediated killing from chronically infected HIV donors. Five days after antibody treatment, cells were used in a quantitative virus outgrowth assay to monitor the number of infectious cells in various treated samples. Humanized antibody panels (A35795, A35796, A35797, 137F2 chimera), and 135C12 (PD) containing heavy and light chain variable CDR loops derived from blocking anti-PD-1 antibodies (for PD-1 / PD-L1 interactions). Humanized antibody panels (A35774, A35783, A35789, A346443, 135C H2L2, 135C H1cL1c, 135C H1cL1b) containing heavy and light chain variable CDR loops derived from non-blocking anti-PD-1 antibodies (of -1 / PD-L1 interactions). , 135C12 chimera) exhibits antibody activity comparable to MK3475 in recovery of HIV peptide-specific CD8 T cell proliferation in functional exhaustion recovery assay. Two antagonist anti-PD-1 antibodies that bind to different epitopes on PD-1 (one blocking (MK3475 or A35795) and non-blocking (A35774) to PD-1 / PD-L1 interactions). The combination enhances the relief of functional exhaustion of HIV-specific CD8 T cells and enhances their proliferation beyond what is achieved with either antibody alone. Two antagonist anti-PD-1 antibodies that bind to different epitopes on PD-1 (one blocking (MK3475) PD-1 / PD-L1 interaction and the other non-blocking (135C H1cL1b or 135C H1cL1c) ) Suppresses the relief of functional exhaustion of HIV-specific CD8 T cells and enhances their proliferation beyond the extent achieved with either antibody alone. Stimulation of the Jurkat PD-1 NFAT reporter cell line with a transiently transfected 293T cell line expressing the TCR activator and PD-L1 protein. A. MK3475 and A35774. B. MK3475 and 135C H1cL1c. Cell surface PD- when either a blocking anti-PD-1 antibody, a non-blocking anti-PD-1 antibody, or a combination of blocking and non-blocking anti-PD-1 antibodies was incubated with memory T cells from a chronically infected HIV donor. Internalization of 1. Cell surface levels of PD-1 in memory CD4 ⁺ T cells (a, c and e) and memory CD8 ⁺ T cells (b, d and f) were measured at various times after antibody treatment. PBMCs from chronically infected HIV donors carrying memory T cells with high basal levels of PD-1 can be treated with blocking anti-PD-1 antibody (MK3475), non-blocking anti-PD-1 antibody (A35774), or blocking and non-blocking. Incubated with any of the anti-PD-1 antibody combinations (A35774 and MK3475) and then stimulated with TCR activator cells in the presence or absence of PD-L1 for 24 hours. In stimulation with TCR activator cells expressing PD-L1, treatment with a combination of blocking and non-blocking anti-PD-1 antibodies resulted in IFN-γ levels (A) and PD-1 ⁺ CD8 T cells in cell medium. Increases relevant intracellular IFN-γ levels (B). Crystals of hPD-1 / Fab A35774 complex protein. A. Heavy and light chain CDR loops of A35774 that interact with human PD-1 protein. B. Modeling of the hPD-1 / Fab A35774 structure using human PD-L1 (PDB 4ZQK) shows that the non-blocking anti-PD-1 antibody binds to a separate, non-overlapping epitope on PD-1 compared to PD-L1. Make sure you do. Evaluate the therapeutic efficacy of a combination of humanized IgG4 non-blocking anti-PD-1 antibody (135C H1cL1c), blocking anti-PD-1 antibody Keytruda® (Keytruda®) (MK3475), or 135C H1cL1c + Keytruda. Therefore, an Invivo efficacy test was performed on PD-1 HuGEMM mice. (A) The average tumor volume of MC38 cells is shown for vehicle control mice in contrast to various antibody treatments. (B) Mean percent inhibition of tumor growth when treated with antibody against vehicle alone control. (C) Percentage of mice in each anti-PD-1 treatment group that showed complete remission of the transplanted MC38 tumor. 137F2 variable weight ( _VH ) chain. 137F2 variable light ( _VL ) chain. 135C variable weight ( _VH ) chain. 135C variable light ( _VL ) chain.

The present disclosure is in vitro and / or in vivo, programmed Cell Death 1 (PD-1) protein on the cell surface (eg, US Pat. No. 5,698,520, incorporated herein by reference in its entirety. The present invention relates to a binding agent that binds to SEQ ID NO: 1, FIG. 1A, FIG. 1B) (eg, human PD-1) of the book (Honjo, et al.). The binder can also bind to isolated PD-1 polypeptides (eg, human PD-1) and / or fragments and / or derivatives thereof, typically in vitro. Further provided are methods of using such binders to diagnose, treat, prevent and / or ameliorate one or more diseases associated with the presence of cells expressing PD-1. For example, the binder may be an antibody capable of reacting with the epitope of PD-1 and / or binding to the epitope of PD-1 (eg, a monoclonal antibody). The "binder" described herein may include, for example, an agonist or antagonist of PD-1. Agonist binding agents are typically binding agents that are unable to restore T cell function and / or expression of PD-1. Agonist PD-1 binders can be useful in treating autoimmune diseases and the like in which PD-1-expressing cells are involved in disease progression. In contrast, antagonist binders are binders capable of restoring T cell function and / or reducing surface expression of PD-1. For example, PD-1 antagonist binders may be able to restore the function of PD-1-expressing T cells from functional exhaustion known to occur in HIV infections and various tumors. Restoration of T cell function can be identified, for example, by measuring proliferation, cytokine production, cytotoxic activity or other characteristics of such cells. Another use of the binders described herein is the selective targeting and elimination of HIV-infected CD4 ⁺ T cell populations, including replicable HIV (eg, latent and / or replicating). PD-1-expressing cells expressing such PD-1 are known to function as major cell reservoirs for replicable HIV. One possible mechanism for eliminating these CD4 ⁺ T cell populations is antibody dependence using the binders described herein (eg, single and / or bispecific PD-1 antibodies). Sexual cell injury (ADCC). In some embodiments, rescue of antigen-specific CD8 ⁺ T cells from functional exhaustion due to PD-1 expression (eg, recovery or amelioration of proliferation, cytokine production and / or cytotoxic activity) of those cells. In order to induce in, for example, one or more PD-1 antagonist binding agents with different specificities (eg, recognizing different epitopes) may be combined. In some embodiments, the binders described herein may be provided for selective elimination and / or suppression of PD-1-expressing cells. In some embodiments, the PD-1 agonists or antagonists described herein are used, for example, to treat infectious diseases (eg, HIV), cancer, and / or particularly autoimmune conditions. It may be used to suppress and / or eliminate PD-1-expressing cells. Other embodiments, uses, etc. are described below.

The binder may be, for example, an antibody such as a monoclonal antibody, which may comprise any one or more (and / or one or more fragments and / or derivatives thereof) of the amino acid sequence shown in Table 1. .. The disclosure further provides the use of such monoclonal antibodies to isolate, identify and / or target cells expressing PD-1. In certain embodiments, these monoclonal antibodies may be reactive to PD-1 expressed on the cell surface. The term "antibody" can mean a total or fragmented antibody in unpurified or partially purified form (eg, hybridoma supernatant, ascites, polyclonal antiserum) or purified form. The antibody may be of any suitable origin or form, including, for example, mice (eg, produced by mouse hybridoma cells), or may be expressed as humanized antibodies, chimeric antibodies, human antibodies and the like. For example, the antibody may be wholly or partially human (eg, IgG (IgG1, IgG2, IgG2a, Ig2b, IgG3, IgG4), IgM, IgA (IgA1 and IgA2), IgD and IgE), dogs (eg, IgGA, etc.). IgGB, IgGC, IgGD), chickens (eg IgA, IgD, IgE, IgG, IgM, IgY), goats (eg IgG), mice (eg IgG, IgD, IgE, IgG, IgM) and / or pigs (eg, IgG, IgD, IgE, IgG, IgM) For example, it may be derived from an IgG, IgD, IgE, IgG, IgM) or rat (eg, IgG, IgD, IgE, IgG, IgM) antibody. Methods of preparing, utilizing and storing various types of antibodies are well known to those of skill in the art and may be suitable for carrying out the present invention (eg, Harlow, et al. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory). , 1988; Hallow, et al. Using Antibodies: A Laboratory Manual, Cold Spring Harbor No. 1, 1998; Kowler and Milstein, Nature, 256: 495 (195). Nature, 321: 522-525 (1986); Richmann et al. Nature, 332: 323-329 (1988); Presta (Curr. Op. Struct. Biol., 2: 593-596 (1992); Verhoeyen et al. (Science, 239: 1534-1536 (1988); Hoogenbo ., J. Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Biol., 222: 581 (1991); Core et al., Monoclonal Antibodies and Cancer , P. 77 (1985); Boerner et al., J. Immunol., 147 (1): 86-95 (1991); Marks et al., Bio / Technology 10, 779-783 (1992); ., Nature 368 856-859 (1994); Morrison, Nature 368 812-13 (1994); Fisher et al., Nature Biotechnology 14, 845-51 (1996); and Huszar, Intern. Rev. Immunol. 13 65-93 (1995); and US Pat. Nos. 4,816,567; 5,545,807; 5,545,806. Documents; the same No. 5,569,825; the same No. 5,625,126; the same No. 5,633,425; the same No. 5,661,016; and the same No. 9 , 090, 994). For certain applications, the antibody may be contained in a hybridoma supernatant or ascites and may be used either directly or after concentration using standard techniques. In other uses, the antibody may be, for example, fractionation by salting and ion exchange chromatography, or protein A, protein G, protein A / G and / or protein L, and / / covalently attached to a solid support such as agarose beads. Alternatively, further purification may be used using affinity chromatography with other ligands. The body may be stored in any suitable form, including frozen formulations (eg, −20 ^o C or −70 ^o C), lyophilized forms, or under normal refrigeration conditions (eg, 4 ° C.). For example, when stored in liquid form, it is preferable to use a suitable buffer such as Tris-buffered saline (TBS) or Phosphate-buffered saline (PBS). In some embodiments, the binder can be prepared as an injectable formulation, such as a non-toxic parenteral ingestible diluent or suspension in a solvent. Suitable vehicles and solvents available include, among others, water, Ringer's solution, and isotonic sodium chloride solution, TBS and / or PBS. Such formulations are suitable for use in vitro or in vivo and can be prepared as known in the art, and the exact formulation will depend on the particular application.

However, the binders described herein are by no means limited to antibodies. For example, the binder may be any compound (eg, a mimic) that exhibits similar binding properties to another. For example, a representative binder may be a binder that binds to PD-1 and / or is capable of competing with a binder (monoclonal antibody) having specificity thereof. In some embodiments, the mimic can exhibit substantially the same affinity in the binding assay with the binder being compared (eg, a monoclonal antibody). Affinity of a particular binder is measured by any suitable assay, such as, but not limited to, FACS staining of endogenous cell surface PD-1 with activated CD4 T cells as described in the Examples. Can be done. One binder has a measured value (eg, apparent binding constant at nanomolar affinity) of about 1-20, 1-5, 5-10, 10-15, 15-20, with each other. Either 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250%. If it is within the range of, it can be said that it has "substantially the same affinity" with another binder. Typical mimetics include, for example, organic compounds that specifically bind to PD-1, or affibodi (Nygren, et al. FEBS J. 275 (11): 2668-76 (2008)), affylin (Ebersbach, etc.). et al. J. Mol. Biol. 372 (1): 172-85 (2007)), Afitin (Krehenbrink, et al. J. Mol. Biol. 383 (5): 1058-68 (2008)), Anticarin (Skerra, A. FEBS J.275 (11): 2677-83 (2008)), Abima (Silverman, et al. Nat. Biotechnol.23 (12): 1556-61 (2005)), DARPin (Stumpp, et). al.Drug Discov.Today 13 (15-16): 695-701 (2008)), Finomer (Grabulovski, et al. J. Biol. Chem. 282 (5): 3196-3204 (2007)), Kunitz domain peptide. (Nixon, et al. Curr. Opin. Drug Discov. Devel. 9 (2): 261-8 (2006)) and / or monobody (Koide, et al. Methods Mol. Biol. 352: 95-109 (2007)). )) And so on. Other mimics include, for example, antibody derivatives such as Fab, _Fab2 , _Fab'single chain antibody, Fv _, single domain antibody, monospecific antibody, bispecific antibody, trispecific. Antibodies, polyvalent antibodies, chimeric antibodies, canine-human chimeric antibodies, canine-mouse chimeric antibodies, antibodies containing canine Fc, humanized antibodies, human antibodies, canineization, CDR grafted antibodies, shark antibodies, nanobody, camel antibodies, micro Examples include body and / or intrabody or derivatives thereof. Other binders are also provided herein, as will be appreciated by those of skill in the art.

Any method known to those of skill in the art may be used to make a binder having (eg, binding) specificity for PD-1. For example, administering one or more PD-1 proteins (eg, PD-1 Fc fusion protein and / or PD-1 His tag protein) to animals such as mice to make and isolate monoclonal antibodies. Can be done. Next, serum (eg, as identified by flow cytometry and / or microscopy) against PD-1 expressed on activated human T lymphocytes to generate an anti-PD-1 hybridoma cell line. Select animals that are reactive. This may be repeated over multiple rounds. For example, the primary criteria for first round binding agent selection were: i) levels of PD-1 staining on activated human T lymphocytes by flow cytometry; (ii) compared to existing anti-PD-1 antibodies. Variety of CDR VH and VL sequences; and PD- pre-bound to (iii) PD-L1 and one of several commercially available anti-PD-1 antibody bindings that bind to different epitopes on PD-1. It may include, but is not limited to, epitope mapping performed by a competitive binding test using one conjugate Luminex bead. Typical first or second round selections are, for example, affinity binding (not a primary criterion as it may not correlate with the stimulatory capacity of anti-PD-1 antibody) and / or binding agents agonists or antagonists. Functional characterization for identification as may also be included.

As described in Example 1 herein, for example, the Exhaustion Functional recovery assay (EFRA) can be used. In this assay, test binders can be assayed for their ability to rescue immune cells such as T cells from exhaustion. This can be identified by measuring the ability of the binding agent to restore proliferation to such cells in the presence of antigens such as test peptides derived from viruses such as the human immunodeficiency virus (HIV). Proliferation is measured in a CFSE assay compared to a control such as test peptide alone, or a positive control such as MK-3475 (pembrolizumab). In some embodiments, if the comparison shows a significant difference (eg, a P-value of <0.001) as compared to the use of the peptide with the peptide alone control or the isotype control mouse IgG1 antibody, binding. The agent is specified to restore growth. This assay is used to identify binders (eg, antibodies) that compete with other binders (eg, PD-L1 or PD-L2) for binding to PD-1 and / or result in functional recovery of immune cells. can do. Example 1 also describes two methods of epitope mapping the antibodies described therein (eg, listed in Table 3) using an assay based on Luminex®. In one biochemical assay, the PD-1 Fc fusion protein is bound to the beads between the anti-PD-1 antibody listed in Table 3 and one of two different commercially available anti-PD-1 antibodies. Then, carry out a competitive binding test. Example 1 is a class of four monoclonal antibodies that bind to distinct epitopes on PD-1: class 1 (competitive with a first monoclonal antibody that blocks the interaction of PD-1 with PD-L1). 2 (competitive with the second monoclonal antibody that binds to PD-1 but does not block the interaction between PD-1 and PD-L1), class 3 (competitive with both the first and second monoclonal antibodies) and class 4 (non-competitive with both the first and second antibodies) is described. In a separate assay, competition for binding to recombinant PD-1 protein was evaluated for the anti-PD-1 antibodies and biotinylated PD-L1 recombinant proteins listed in Table 3. Antibodies that induced proliferation in EFRA were identified from all four binding classes proposed to bind to different epitopes on PD-1. In addition, EFRA is an anti-PD-1 antibody that is either blocking or non-blocking to PD-1 / PD-L1 interactions and specifically restores proliferative function to HIV-specific CD8 ⁺ T cells. Allowed identification.

Binder combinations can also be identified. In some embodiments, combinations can be identified that result in a statistically significant difference compared to the results obtained with only one or more binders and no other substances. In some embodiments, combinations can be identified that exhibit a synergistic or collaborative ability to restore immune cell function. In some embodiments, the combination comprises a first binder that blocks the interaction of PD-1 with PD-L1 along with a second binder that does not block the interaction between PD-1 and PD-L1. You can go out. The first and second binders may be different components, such as, for example, two or more different monoclonal antibodies or derivatives thereof, or, for example, a bifunctional antibody (single antibody having multiple binding specificities). Or a derivative thereof) may be found on the same component. For example, a typical bifunctional antibody has a first binding region that blocks the interaction between PD-1 and PD-L1 and a second binding region that does not block the interaction between PD-1 and PD-L1. May include. In addition, combinations are also contemplated to provide multiple types of each binder. For example, the combination comprises one or more binders that do not block the interaction between PD-1 and PD-L1 as well as multiple types of binders that block the interaction between PD-1 and PD-L1. You can go out. In some embodiments, the combination is one or more bindings that block the interaction between PD-1 and PD-L1 with the plurality of binders that do not block the interaction between PD-1 and PD-L1. May contain agents. In some embodiments, the combination comprises a plurality of binders that block the interaction of PD-1 with PD-L1 together with a plurality of binders that do not block the interaction of PD-1 with PD-L1. You may be. Such combinations described herein may be combined with one or more other agents that may affect immune cell function, such as antibodies to CTLA-4 and the like. Those skilled in the art will appreciate that a number of such combinations may be suitable for the applications described herein.

If the binder is an antibody, the antibody can be identified with respect to the nucleotide and / or amino acid sequences corresponding to its variable regions and / or complementarity determining regions (“CDR”). The variable region / CDR sequence can be used in combination with one or more other variable region / CDR amino acid sequences. The variable region / CDR amino acid sequence can instead and / or further be flanked by one or more types of constant region polypeptides of the antibody molecule. For example, the CDR amino acid sequences shown in Tables 1A and 1B are antibody subtypes from which and / or their CDR amino acid sequences of any antibody molecule of the same or different species (eg, human, goat, rat, sheep, chicken) are derived. May be adjacent to or coupled to the constant region of. For example, exemplary binding agents may be monoclonal antibodies produced by the listed hybridomas, or those derived from or related thereto, and / or shown in Tables 3 and 11-13. Any of the amino acid sequences of SEQ ID NOs: 1-190 and / or the amino acid sequences shown in Tables 1A and 1B, which may have substantially the same affinity and / or proliferative effect and / or have the same binding class. It may have one or more. The binder may comprise an antibody heavy chain and / or a light chain, each containing one or more constant and / or variable regions. The variable region typically comprises one or more CDRs capable of determining the binding specificity of the antibody. Monoclonal antibodies can be identified by analysis of the amino acid sequences of such variable regions (eg, which can be encoded by such nucleotide sequences). For example, the typical amino acid sequence of the heavy chain CDR of the binding agent that binds to PD-1 is shown in at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 1-190 and / or Tables 1A and / or 1B. It may contain one or more including any other sequence. Any other variable region and / or any other variable region and / or any of the amino acid sequences described herein and / or any fragment and / or derivative thereof, in any order and / or combination, using standard techniques. It may be combined with the CDR to form a hybrid and / or fusion binder and / or inserted into other heavy and / or light chain variable regions. As a typical combination of CDRs found within the PD-1 (eg, human PD-1) binders of the present disclosure (eg, combinations of heavy and / or light chain CDR1, CDR2 and CDR3 amino acid sequences), eg, Examples include the embodiments shown in Tables 1A and / or 1B.

In a preferred embodiment, the heavy chain CDRs of each clone are combined with their respective light chain CDRs to form a binder. In some embodiments, the binder may include heavy chain CDRs and light chain CDRs shown below:
122F10 (SEQ ID NOs: 1, 24, 47, 70, 93, and 116);
139D6 (SEQ ID NOs: 2, 25, 48, 71, 94, and 117);
135D1 (SEQ ID NOs: 3, 26, 49, 72, 95, and 118);
134D2 (SEQ ID NOs: 4, 27, 50, 73, 96, and 119);
121G1 (SEQ ID NOs: 5, 28, 51, 74, 97, and 120);
136B5 (SEQ ID NOs: 6, 29, 52, 75, 98, and 121);
127C2 (SEQ ID NOs: 7, 30, 53, 76, 99, and 122);
137F2 (SEQ ID NOs: 8, 31, 54, 77, 100, and 123);
138H5 (SEQ ID NOs: 9, 32, 55, 78, 101, and 124);
140A1 (SEQ ID NOs: 10, 33, 56, 79, 102, and 125);
135H12 (SEQ ID NOs: 11, 34, 57, 80, 103, and 126);
131D11 (SEQ ID NOs: 12, 35, 58, 81, 104, and 127);
132F7 (SEQ ID NOs: 13, 36, 59, 82, 105, and 128);
126E4 (SEQ ID NOs: 14, 37, 60, 83, 106, and 129);
135G1 (SEQ ID NOs: 15, 38, 61, 84, 107, and 130);
136E10 (SEQ ID NOs: 16, 39, 62, 85, 108, and 131);
135C12 (SEQ ID NOs: 17, 40, 63, 86, 109, and 132);
136F4 (SEQ ID NOs: 18, 41, 64, 87, 110, and 133);
136B4 (SEQ ID NOs: 19, 42, 65, 88, 111, and 134);
135E10 (SEQ ID NOs: 20, 43, 66, 89, 112, and 135);
140G5 (SEQ ID NOs: 21, 44, 67, 90, 113, and 136);
122H2 (SEQ ID NOs: 22, 45, 68, 91, 114, and 137); or 139F11 (SEQ ID NOs: 23, 46, 69, 92, 115, and 138).

Exemplary heavy and light chain variable regions comprising one or more of the CDRs described herein may include, but are not limited to, humanized heavy and / or light chain variable regions. For example, exemplary humanized "137F2" variable heavy chain sequences comprising 137F double heavy chain CDRs (SEQ ID NOs: 8, 31 and 54) include:

QMQLVQSGPEVKKPGTSVKVSCKASGFFTNYWIGWVRQAPGQUAREWMGDIYPGGGYTNYNEKFKGRVTITADKSTSTAYMELSSLDTAVYYCARGYDFVLDRWGQGTTVSS (A355YDFVLDRWGQGTTVSS)

QMQLVQSGPEVKKPGTSVKVSCKASGFFTNYWIGWVRQAPGQUAREWMGDIYPGGGYTNYNEKFKGRVTITADKSTSTAYMELSSLDTAVYYCARGYDFVLDRWGQGTTVSS (A3596)

QMQLVQSGPEVKKPGTSVKVSCKASGFFTNYWIGWVRQAPGQUAREWMGDIYPGGGYTNYNEKFKGRVTITADKSTSTAYMELSSLDTAVYYCARGYDFVLDRWGQGTTVSS (A35793)

QMQLVQSGPEVKKPGTSVKVSCKASGFFTNYWIGWVRQAPGQUAREWMGDIYPGGGYTNYNEKFKGRVTITADKSTSTAYMELSSLDTAVYYCARGYDFVLDRWGQGTTVSS (A3518)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWIGWVRQUAPGQGLEWMGDIYPGGGYTNYNEKFKGRVTITADKSTSTAYMELSSLSREDTAVYYCARGYDFVLDRWGQGTTVSS (35)

EVQLVQSGAEVKKHGESLKISCKGSGYSFTNYWIGWVRQATGQGLEWMGDIYPGGGYTNYNEKFKGRVTITADKSTSTAYMELSSLSREDTAVYYCARGYDFVLDRWGQGTTVSS7

QMQLVQSGAEVKKTGSSVKVSCKASGYTFTNYWIGWVRQMPGKGLEWMGDIYPGGGYTNYNEKFKGRVTITADKSTSTAYMELSSLSREDTAVYYCARGYDFVLDRWGQGTTVSS7

QVQLVQSGSELKKPGASVKVSCKASGYTFTNYWIGWVRQAPGKGLEWMGDIYPGGGYTNYNEKFKGRVTITADKSTSTAYMELSSLSREDTAVYYCARGYDFVLDRWGQGTTVSS

QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWIGWVRQAPGQGLEWMGDIYPGGGYTNYNEKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYDFVLDRWGQGTVSS1

QVQLVQSGAEVKKPGASVKVSCKAAGYTFTNYWIGWVRQAPGQGLEWMGDIYPGGGYTNYNEKFKGRVTMTADTSTSTVYMELSSLRSEDTAVYYCARGYDFVLDRWGQGTLVTVSS1

QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWIGWVRQAPGQGLEWIGDIYPGGGYTNYNEKFKGRVTMTDTSTSTVYMELSSLDTAVYYCARGYDFVLDRWGQGTVTVSS9 (1)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWIGWIRQAPGQGLEWIGDIYPGGGYTNYNEKFKGRATTLTADTSTSTVYMEVSSLRSEDITAVYYCARGYDFVLDRWGQGTLVTVSS (137F)

Exemplary humanized "137F2" variable light chain sequences comprising 137F2 light chain CDRs (SEQ ID NOs: 77, 100 and 123) include:

DVMTQSPAFLSVTPGEKVTITCKSSQSLFNSETQKNYLAWYQQKPGQPPPKLLIYWASTRESGVPSRFSGSGSGTDFTLTSSLQPDFATTYCKQSYTLRTFGGTKLEIK (A35790)

DVMTQSPAFLSVTPGEKVTITCKSSQSLFNSETQKNYLAWYQQKPGQPPPKLLIYWASTRESGVPSRFSGSGSGTDFTLTSSLQPDFATTYCKQSYTLRTFGGTKLEIK (A3596-)

DVMTQSPAFLSVTPGEKVTITCKSSQSLFNSETQKNYLAWYQQKPGQPPPKLLIYWASTRESGVPSRFSGSGSGTDFTFTISLEAEDAAATTYCKQSYTLRTFGGTKLEIK (A3573V)

DVMTQSPAFLSVTPGEKVTITCKSSQSLFNSETQKNYLAWYQQKPGQAPRLLLYWASTRESGVPSRFSGSGSGTDFTFTISLEAEDAAATTYCKQSYTLRTFGGTKLEIK (A35518-V)

DVMTQSPAFLSVTPGEKVTITCKSSQSLFNSETQKNYLAWYQQKPGQAPRLLLIYWASTRESGVPSRFSGSGSGTDFTLTSSLQPDFATTYCKQSYTLRTFGGTKLEIK (A355)

DIQMTQSLSLSVSVGDRVTITCKSSQSLFNSETQKNYLAWYQQKPGQPQPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSSLQPDFATTYCKQSYTLRTFGGTKLEIK (A35797-V)

DVMTQSPAFLSVTPGEKVTITCKSSQSLFNSETQKNYLAWYQQKPGQPPPKLLIYWASTRESGVPSRFSGSGSGTDFTLTSSLQPDFATTYCKQSYTLRTFGGTKLEIK (A355)

DVMTQSPAFLSVTPGEKVTITCKSSQSLFNSETQKNYLAWYQQKPGQPPPKLLIYWASTRESGVPSRFSGSGSGTDFTFTISLEAEDAAATTYCKQSYTLRTFGGGTKLEIK (A35805-VL)

DIVMTQSPDSLAVSLGERATINCKSSQSLFNSETQKNYLAWYQQKPGQPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSSLQAEDVAVYYCKQSYTLRTFGQGTKLEIK (137F VL1)

DIVMTQSPDSLAVSLGERATTITCKSSQSLFNSETQKNYLAWYQQKPGQPKLLIFWASTRESGVPDRFLGSGSGSGTDFTLSSLQAEDVAVYYCKQSYTLRTFGQGTKLEIK (137F VL2)


DIVMTSPDSLAVSLGERATINCKSSQSLFNSETQKNYLAWYQQKPGQPKLLLIYWASTRESGVPDRFSGSGSGTDFTLTISSSLQAEDVAVYYCKQSYTLRTFGQGTKLEIK (137F VL1b)

DIVMTQSPDSLAVSLGERATTITCKSSQSLFNSETQKNYLAWYQQKPGQPKLLIFWASTRESGVPDRFSGSGSGTDFTLTISSSLQAEDVAVYYCKQSYTLRTFGQGTKLEIK (137F VL2)

DIVMTQSPDSLAVSLGERATMTCKSSQSLFNSETQKNYLAWYQQKPGQPKLLIFWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCKQSYTLRTFGQGTKLEIK (137F VL1).

Exemplary humanized "135C12" variable heavy chain sequences comprising 35C12 heavy chain CDRs (SEQ ID NOs: 17, 40 and 63) include:

EVQLVQSGAEVKKPGESSLKISCKGSGYSFTNFYIHWVRQAPGQRLEWMGSIYPNYGDTAYNQKFKDRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARGYSYAMDYWG.

EVQLVQSGAEVKKPGESSLKISCKGSGYSFTNFYIHWVRQARGQRLEWIGSIYPNYGDTAYNQKFKDRFVFSLDTSVSTAYLQISSSLKAEDTAVYYCARGYSYAMDYWGQGTTVS.

EVQLVQSGAEVKKPGESSLKISCKGSGYSFTNFYIHWVRQAPGQRLEWMGSIYPNYGDTAYNQKFKDRFVFSLDTVSTAYLQISSLKAEDTAVYYCARGYSYAMDYWG.

EVQLVQSGAEVKKPGBATVKISCCKVSGYTFTNFYIHWVRQARGQRLEWIGSIYPNYGDTAYNQKFKDRVTITADKSTSTAYMERSSLDTAVYYCARGYSYAMDYWGQGTTVSS (A3663)

EVQLVQSGAEVKKPGBATVKISCCKVSGYTFTNFYIHWVRQAPGKGLEWMGSIYPNYGDTAYNQKFKDRVTITADKSTSTAYMELSSLDTAVYYCARGYSYAMDYWGQGTTVSS (A35)

EVQLVQSGAEVKKPGESSLKISCKGSGYSFTNFYIHWVRQMPGKGLEWMGSIYPNYGDTAYNQKFKDRVTITADKSTSTAYMELSSLDTAVYYCARGYSYAMDYWGQGTTVSS9

EVQLVQSGAEVKKPGESSLKISCKGSGYSFTNFYIHWVRQMPGKGLEWMGSIYPNYGDTAYNQKFKDRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARGYSYAMDYWGQGTTVS

EVQLVQSGAEVKKPGESSLKISCKGSGYSFTNFYIHWVRQMPGKGLEWMGSIYPNYGDTAYNQKFKDRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARGYSYAMDYWGQGTTV1

QVQLVQSGAEVKKPGASVKVSCKASGYTFTNFYIHWVRQAPGQGLEWMGSIYPNYGDTAYNQKFKDRVTMTRDTSTSTVYMELLSSLDTAVYYCARGYSYAMDYWGQGT

QVQLVQSGAEVKKPGASVKVSCKASGYTFTNFYIHWVRQAPGQGLEWMGSIYPNYGDTAYNQKFKDRVTMTVDKSTSTVYMELLRSLRSEDTAVYYCARGYSYAMDYWGQGT

QVQLVQSGAEVKKPGASVKVSCKASGYTFTNFYIHWVKQAHGQGLEWMGSIYPNYGDTAYNQKFKDRVTMTVDKSTSTVYMELLRSLRSEDTAVYYCARGYSYAMDYWGQGT

QVQLVQSGAEVKKPGASVKVSCKASGYTFTNFYIHWVRQAPGQGLEWIGSIYPNYGDTAYNQKFKDRVTMTVDTSTSTVYMELSSLDTAVYYCARGYSYAMDYWGQGTLVTVSS1 (135C)

QVQLVQSGAEVKKPGASVKMSCKASGYTFTNFYIHWVRQUAPGQGLEWIGSIYPNYGDTAYNQKFKDRATLTVDTSTSTAYMELSSLDTAVYYCARGYSYAMDYWWGQGTLVTVSS (135C)

QVQLVQSGAEVKKPGASVKMSCKASGYTFTNFYIHWVRQAPGQGLEWIGSIYPNYGDTAYNQKFKDRATLTVDKSTSTAYMELSSLDTAVYYCARGYSYAMDYWGQGTLVTVSS1 (135C)

Exemplary humanized "135C12" variable light chain sequences comprising the 135C12 light chain CDRs (SEQ ID NOs: 86, 109 and 132) include:

DIQMTQSPSVSSASVGDRVTITCSASSQGISGDLNWYQQKPGQAPRLLLYHTSSLHSGVPSRFSGSGSGTDFTLTISSSLQPEDFATYYCQYYSKDLLTFGGTKLEIK (A35775-VL)

DIQMTQSLSLSVSVGDRVTITCSASSQGISGDLNWYQQKPGKTPKLLIYHTSSSLHSGVPSRFSGSGSGSTDFTSSLQPEDFATYYCQYYSKDLLTFGGTKLEIK (A35783-VL) (SEQ ID NO: 17)

DIQMTQSPSVSSASVGDRVTITCSASSQGISGDLNWYQQKPGKAPKLLIYHTSSSLHSGVPSRFSGSGSGTDFTLTISSSLQPEDFATYYCQYYSKDLLTFGGTKLEIK (A35774-VL)

DIQMTQSPSVSSASVGDRVTITCSASSQGISGDLNWYQQKPGQAPRLLLIYHTSSSLHSGVPSRFSGSGSGTEFTLTISRRLEPEDFAVYYCQYYSKDLLTFGGTKLEIK (A36443-VL) (A36443-VL)

DIQMTQSPSTLSASVGDRVTITCSASSQGISGDLNWYQQKPGQAPRLLLYHTSSLHSGVPSRFSGSGSGTDFTLTISSSLQPEDFATYYCQYYSKDLTLTFGGGTKLEIK (A35777-VL) (A35777-VL)

DIQMTQSPSVSSASVGDRVTITCSASSQGISGDLNWYQQKPGQAPRLLLIYHTSSSLHSGIPARFSGSGSGTDFTLTISRLEPEDFAVYYCQYYSKDLLTFGGTKLEIK (A35789-VL) (A35789-VL)

DIQMTQSSPSTLSVGDRVTITCSASSQGISGDLNWYQQKPGKTPKLLIYHTSSSLHSGVPSRFSGSGSGTDFTLTISSSLQPEDFATYYCQYYSKDLLTFGGTKLEIK (A36448-VL) (A36448-VL)

DIQMTQSSPSTLSVGDRVTITCSASSQGISGDLNWYQQKPGQAPRLLLYHTSSLHSGIPARFSGSGSGTDFTLTISSSLQPEDVABYYCQYYSKDLLTFGGTKLEIK (A36437-VL) (A36437-VL)

DIQMTQSLSLSVSVGDRVTITCSASSQGISGDLNWYQQKPGKAPKLLIYHTSSSLHSGVPSRFSGSGSGSTDFTSSLQPEDFATYYCQYYSKDLLTFGGTKLEIK (135C VL1) (SEQ ID NO: 18)

DIQMTQSLSLSVSVGDRVTITCSASSQGISGDLNWYQQKPGKAVKLLIYHTSSSLHSGVPLRFSGSGSGTDYTLTISSSLQPEDFATYYCQYYSKDLLTFGGTKLEIK (135C VL2)

DIQMTQSLSLSVSVGDRVTITCSASSQGISGDLNWYQQKSDGAVKLLIYHTSSSLHSGVPLRFSGSGSGTDYTLTISSSLQPEDFATYYCQYYSKDLLTFGGTKLEIK (135C VL3)

DIQMTQSLSLSVSVGDRVTITCSASSQGISGDLNWYQQKPGKAPKLLIYHTSSSLHSGVPSRFSGSGSGSTDYTLTISSSLQPEDFATYYCQYYSKDLLTFGGTKYYCQYYSKDLLTFGGTKLEIK (135C VL1b)

DIQMTQSLSLSVSVGDRVTITCSASSQGISGDLNWYQQKPGKAVKLLIYHTSSSLHSGVPSRFSGSGSGTDYTLTISSSLQPEDFATYYCQYYSKDLLTFGGTKLELK (135C VL1c).

Exemplary nucleic acid sequences encoding these amino acid sequences and / or methods for identifying them are also described herein (eg, SEQ ID NOs: 191-243). Any of the compatible humanized variable sequences described above or herein may be combined with each other to provide a binder such as an antibody that binds PD-1. For example, in certain embodiments, SEQ ID NOs: 143 and 155 may be combined into a binder (eg, like monoclonal antibody (mAb) A35795); SEQ ID NOs: 166 and 180 may be combined into a binder (eg, mAb A35774). (As in); SEQ ID NOs: 196 and 189 may be combined into a binder (eg, mAb 135C H1cL1b); SEQ ID NOs: 196 and 190 may be combined into a binder (eg, mAb 135C H1cL1c). .. Further, as shown in Table 2, variable heavy chains and light chains may be present (eg, bound) within the binder:

The "blocking" antibody is to PD-1 on the cell surface, which is identified by any suitable assay, such as, but not limited to, Luminex biochemical PD-1 / PD-L1 interaction assay (FIG. 1). It competes with PD-L1 in terms of binding. Epitope mapping tests performed with blocking anti-PD-1 antibody locate regions of PD-1 that overlap with PD-1 / PD-L1 interactions. "Non-blocking" antibodies are partially, but not limited to, PD-L1 with respect to binding to PD-1 in suitable assays such as, for example, Luminex biochemical PD-1 / PD-L1 interaction assay (FIG. 1). It only competes with or does not compete at all. Epitope mapping tests and / or human PD-1 / _Fab co-crystallization tests confirm that non-blocking antibodies do not bind PD-1 at sites that overlap with PD-1 / PD-L1 interactions. In certain embodiments, the binder may contain, for example, two or more pairs of such sequences in a bispecific antibody (eg, in combination with SEQ ID NOs: 143 and 155, and SEQ ID NOs: 166 and 180). It may be a binder or a combination of binders). Other combinations may also be useful, as confirmed by those of skill in the art.

Binders comprising the CDRs of Tables 1A and / or 1B, or the binders described therein (eg, comprising SEQ ID NOs: 139-190 and / or shown in Table 2), have the following characteristics: Can be shown:

As described in the Examples, the epitope mapping test is designated as "P1" and "P2", at least two conserved patches on PD-1 to which the binders described therein can bind. Shown (including linear and / or conformational epitopes) (see, eg, FIGS. 11a and 11b). The P1 patch is evolutionarily conserved and corresponds to the central region of PD-1 involved in the interaction between PD-1 and the PD-L1 / PD-L2 ligand, purple in FIGS. 2 and 11a. Corresponds to the circle of. The second "patch" P2 is also evolutionarily conserved, occupying the same surface region as the P1 patch but with a different amino acid sequence (FIG. 11b). P2 does not have an already identified structural or functional role in PD-1. For example, 135C12 (SEQ ID NOs: 17, 40, 63, 86, 109, and 132), 139D6 (SEQ ID NOs: 2, 25, 48, 71, 94, and 117), 135D1 (SEQ ID NOs: 3, 26, 49, 72, 95). , And 118), and the PD-1 binding agents described herein, such as anti-PD-1 antibodies comprising the amino acid sequences of 136B4 (SEQ ID NOs: 19, 42, 65, 88, 111, and 134). It binds to an epitope that overlaps the P2 patch, thereby providing direct evidence of the functional importance of this newly identified functional region of PD-1. Examples describe epitope mapping tests performed with the modified PD-1 polypeptide shown in FIG. 3 and SEQ ID NOs: 244-274. In these tests, the non-blocking antibody with the highest "functional capacity" or "antagonist activity" was the region of the M4 amino acid substitution (serine 38 to alanine, proline 39 to alanine, and leucine 41 to alanine (Figure). 3; SEQ ID NO: 247)) and / or M17 amino acid substitution region (asparagine 102 to alanine and arginine 104 to alanine (FIG. 3; SEQ ID NO: 260)) and / or M18 amino acid substitution region (aspartic acid). 105 to alanine (FIG. 3; SEQ ID NO: 261)) and / or the region of the M26 amino acid substitution (arginine 138 to alanine and glutamate 141 to alanine (FIG. 3; SEQ ID NO: 269)) and / or the M31 amino acid. It was shown to bind to a "patch" of PD-1 that overlaps the region of substitution (leucine 41 to alanine and valine 43 to leucine (FIG. 3; SEQ ID NO: 274)). This "patch" is referred to herein as "P2". These tests demonstrate that P2 comprises at least one epitope to which such a non-blocking antibody binds, including linear epitopes, conformational epitopes, or combinations thereof. Given that this region has no conventional involvement in the functional activity of PD-1, binding to P2 is a novel action at a novel site on PD-1 where antagonist activity to PD-1 can be exerted. It is proposed here to show the mechanism. Other antibodies, antibody fragments, or other protein binding agents capable of interacting with the P2 region of PD-1 are also distinguished from anti-PD-1 antibodies that act through blocking of PD-1 / PD-L1 interactions. It is further proposed that it may act as a PD-1 antagonist in a manner complementary to it. The binders described therein interact, for example, with ligands that have not yet been identified; interfere with, induce and / or enhance PD-1 multimerization; and / or P2. By interacting with (eg, binding) with, it may alter intracellular signaling associated with PD-1; Thus, the binders described therein that interact with (eg, bind to) P2 may provide PD-1 antagonist function via these mechanisms or any other mechanism that should still be identified.

Accordingly, the present disclosure provides a method of affecting the function of PD-1 by interacting with this P2 patch of PD-1 intracellularly or on the cell. The amino acid residues in the P2 patch of PD-1 are threonine 36, phenylalanine 37, serine 38, proline 39, leucine 41, valine 43, alanine 50, threonine 51, phenylalanine 52, threonine 53, cysteine 54, serine 55, aspartic acid. 102, arginine 104, aspartic acid (aspartate) 105, phenylalanine 106, histidine 107, methionine 108, arginine 138 and glutamic acid (glutamate) 141 may be included, where the amino acid numbering corresponds to SEQ ID NO: 275. do. In certain embodiments, the amino acid residues of the P2 patch are threonine 36, phenylalanine 37, alanine 50, threonine 51, phenylalanine 52, threonine 53, cysteine 54, serine 55, aspartic acid (aspartate) 105, phenylalanine 106, histidine. 107 and methionine 108 may be included, where the amino acid numbering corresponds to SEQ ID NO: 275. In certain embodiments, the amino acid residues of the P2 patch may comprise the amino acid residues serine 38, proline 39, leucine 41, valine 43, asparagine 102, arginine 104, and / or aspartic acid (aspartate) 105. .. Thus, in certain embodiments, the method comprises the amino acids threonine 36, phenylalanine 37, serine 38, proline 39, leucine 41, valine 43, alanine 50, treonine 51, phenylalanine 52, treonine 53, cysteine 54, serine 55, aspartic acid 102, It may include interacting with arginine 104, aspartic acid (aspartate) 105, phenylalanine 106, histidine 107, and / or methionine 108, where the amino acid numbering corresponds to SEQ ID NO: 275. In certain embodiments, the method comprises the amino acids threonine 36, phenylalanine 37, alanine 50, threonine 51, phenylalanine 52, treonine 53, cysteine 54, serine 55, aspartic acid (aspartate) 105, phenylalanine 106, histidine 107 and /. Alternatively, it may include interacting with methionine 108. In certain embodiments, the method may include interacting with the amino acids serine 38, proline 39, leucine 41, valine 43, aspartin 102, arginine 104, and / or aspartic acid (aspartate) 105. In certain embodiments, the method comprises interacting with any amino acid within the P2 patch (eg, the portion of PD-1 containing the amino acid residues described above) and / or overlapping with the P2 patch. In certain embodiments of such a method, the method comprises one or more amino acid residues corresponding to serine 38, proline 39, and / or leucine 41 of SEQ ID NO: 275 (M4; FIG. 3; SEQ ID NO: 247). And / or one or more amino acid residues corresponding to aspartin 102 and / or arginine 104 in relation to SEQ ID NO: 275 (M17; FIG. 3; SEQ ID NO: 260); and / or in relation to SEQ ID NO: 204. One or more amino acid residues corresponding to aspartic acid (aspartate) 105 (M18; FIG. 3; SEQ ID NO: 261); and / or one or more corresponding to arginine 138 and / or glutamic acid (glutamate) 141 or Interact with multiple amino acid residues (M26; FIG. 3; SEQ ID NO: 269) and / or one or more amino acid residues corresponding to leucine 41 and / or valine 43 (M31; FIG. 3; SEQ ID NO: 274). Including that. In certain embodiments, the interaction can be reduced and / or eliminated by modifying (eg, substituting, deleting) any one or more of such amino acid residues. In certain embodiments, the method comprises an antagonistically affecting the function of PD-1. In certain embodiments, the method comprises interacting with PD-1 using a PD-1 binder. In certain embodiments, the PD-1 binding agent is, for example, one or more of the amino acid residues corresponding to leucine 41 and / or valine 43 (M4) of SEQ ID NO: 275; and / or in connection with SEQ ID NO: 275. It has specificity in a region (eg, an epitope) containing one or more amino acid residues corresponding to asparagine 102 and / or arginine 104 (M17). In certain embodiments, the interaction can be caused by a binder capable of binding PD-1 (SEQ ID NO: 275) but not PD-1 M4 (SEQ ID NO: 247) and / or PD-1 (. It can be caused by a binder that has the ability to bind to SEQ ID NO: 275) but not to PD-1 M17 (SEQ ID NO: 260). In certain embodiments, the method comprises interacting with PD-1 at sites (eg, P1 patches) and P2 involved in the interaction of PD-1 with PD-L1 and / or PD-L2. You can stay. The "one or more amino acid residues corresponding to" the amino acid "corresponding to the amino acid" of SEQ ID NO: 275 "is an amino acid in another version of PD-1 at a position similar to that found in SEQ ID NO: 275 (FIG. 3H). Is referred to. However, one of ordinary skill in the art will assume that an amino acid in a PD-1 polypeptide other than SEQ ID NO: 275 (FIG. 3H) "corresponds" to a particular amino acid in SEQ ID NO: 275 (FIG. 3H), depending on its context within the polypeptide. You will understand that it can be identified. For example, monkey PD-1 (SEQ ID NO: 276 (FIG. 3I)) contains leucine at position 41, similar to SEQ ID NO: 275. However, another PD-1 numbering is, for example, one or more additions, deletions and / or such that the "corresponding" leucine can be found, for example, at position 40 or 43 in that particular PD-1. It may be different because of the substitution. However, the leucine "corresponds" to leucine 41 in relation to its context within SEQ ID NO: 275 (FIG. 3H, eg, it is surrounded by the amino acids PA and LV as well as SEQ ID NO: 275 (FIG. 3H)). Will be understood by those skilled in the art. In certain embodiments, the binder is the amino acids F37, P39, A40, L41, V43, L138, R139, and R143; and / or the amino acids P34 of PD-1 (SEQ ID NO: 275). , S137, and R139 (eg, A35774 is F37, P39, A40, L41, V43, L138, R139 and R143 for _VH CDR loops, and P34, E136, S137 and R139 for _VL CDR loops. including). As will be appreciated by those skilled in the art, such methods and other embodiments of amino acids (eg, those "corresponding" to another) are also contemplated herein.

Binding affinity can be identified by any technique available to those of skill in the art. The binding affinity data shown in Table 3 was evaluated by flow cytometric staining of endogenous cell surface PD-1 on CD4 T cells stimulated with phytohaemagglutinin (PHA) for 3-6 days. Binding classes can also be identified by any technique available to those of skill in the art. The binding class data presented in Table 3 were identified by the Luminex assay competitive binding test. In Table 3, the binding class 1 monoclonal antibody is an antibody identified as competing with the EH12.2H7 clone commercial antibody (available from BioLegend, San Diego, CA (eg, product number 329905)); Class 2 antibodies are antibodies identified as competing with J116 clone commercial antibodies (available from Affymetrix eBioscience, San Diego, CA (eg, product numbers 16-9989-80)); The antibody is an antibody identified as competitive with both EH12.2H7 and J116 antibodies; and Class 4 monoclonal antibody cloned antibodies are PD- in the presence of both EH12.2H7 and J116 antibodies. An antibody identified as binding to 1.

Proliferation effects can be identified by any technique available to those of skill in the art. For example, the EFRA system described above and used in Example 1 can be used. Such assays were used to identify the proliferative effect data shown in Table 3. Briefly, carboxyfluoresceinsk, which isolates peripheral blood mononuclear cells (PBMCs) from chronic HIV-infected subjects and stimulates the cells with HIV-specific peptides in the presence and absence of anti-PD-1 antibody. It is a cynimidyl ester (CFSE) assay. Also referred to as a control anti-PD1 antibody (Merck antibody MK-3475, pembrolizumab or Keytruda® (US Pat. Nos. 8, 354, 509, and 8, 900, 587). (Available from Merck &CO.); And / or RASKGVSTSGYSYLH (SEQ ID NO: 287), LASYLES (SEQ ID NO: 288), QHSRDLPLT (SEQ ID NO: 289), NYMY (SEQ ID NO: 290), GINPSNGGTNFNEKFKN (SEQ ID NO: 291), and RDYRFDMGFDY (including SEQ ID NO: 292))) was also tested as a positive control. After 6 days of incubation, HIV-specific CD8 T cell proliferation was evaluated in anti-PD-1 treated samples compared to peptide-only controls and expressed as a percentage above the control (proliferative effect).

In some embodiments, techniques used to identify and characterize PD-1 binders, such as antibodies, are combined to provide a system for identifying and characterizing such binders. Can be done. Candidates to restore function to immune cells, such as those measured by proliferation of one or more candidate binders, such as one or more monoclonal antibodies, in the presence of immunogenic peptides, etc. To identify the ability of the binder, it can be assayed by EFRA or a similar assay. In some embodiments, this type of assay can be used as the first screen to ensure that candidate binders to be further investigated can restore immune cell function. In some embodiments, these types of assays can be followed by an assay that identifies binding affinity for immune cells, such as activated peripheral blood mononuclear cells (PBMCs). In some embodiments, the assay may use techniques such as FACS (fluorescence activated cell sorting). In some embodiments, the assay comprises a competitive binding test using known binding agents such as the presence or absence of non-specific binding and / or an anti-PD1 antibody (eg, Merck antibody MK-3475). good. Following these assays, CDR sequencing of candidate binders, such as those provided in Tables 1A and / or 1B above, may be performed. The EFRA, affinity identification, epitope mapping test, and CDR identification methods described therein together provide a system in which candidate binders can be identified.

Any of the amino acid sequences of Tables 1A and / or 1B and / or any of SEQ ID NOs: 139-190 (and / or any one or more fragments and / or derivatives thereof) are further desired by those of skill in the art. It may be replaced with any other amino acid. For example, one of ordinary skill in the art can make a conservative substitution by substituting a particular amino acid with another amino acid shown in Table 4 below. The particular amino acid substitution selected may depend on the location of the site selected. Such conservative amino acid substitutions have little or no effect on the size, polarity, charge, hydrophobicity or hydrophilicity of the amino acid residue at that position, and in particular result in a reduction in PD-1 binding. It may include substitution of a natural amino acid residue with an unnatural residue so as not to cause it.

Therefore, conservative or non-conservative amino acid substitutions in the amino acid sequences of the binders described therein are also contemplated. For example, amino acid substitutions corresponding to the amino acid sequences of the humanized variable weight ( _VH ) chain and the light ( _VL ) chain described therein (eg, SEQ ID NOs: 139 to 190) may be performed. Exemplary non-conservative and conservative substitutions (the same can be described using Table 4) are shown in Tables 5-8 below (see also FIGS. 25A-D):

In some embodiments, the present disclosure is a binding agent having multiple specificities such that a single binding agent can bind to PD-1 and at least one other second antigen (eg, a cell surface protein). I will provide a. In some embodiments, the second antigen may be an antigen expressed by cells infected with an infectious agent. For example, the typical second antigen may be the HIV Env antigen. Such binders can bind to the second antigen and / or function to neutralize the infectious agent. In certain embodiments, it is a bispecific binder having bispecificity to, for example, PD-1 and HIV antigens such as Env and / or other antigens. The HIV immunogen may be derived from any of the subtypes described herein or any other subtype. In some embodiments, such binding agents are: PD-1 agonist / Env binding; PD-1 agonist PD-1 / Env binding and neutralization; PD-1 antagonist / Env binding; and / or PD-1. It may include antagonist / PD-1 / Env binding and neutralization. Given the prevalence of the various subtypes, it may be preferable to select antigens derived from HIV-1 subtypes B and / or C. Also, in a single composition, from multiple HIV subtypes (eg, HIV-1 subtypes B and C, HIV-2 subtypes A and B, or a combination of HIV-1 and HIV-2 subtypes). It may be desirable to include a binding agent that is specific to the antigen. Multiple PD-1 specificities (eg, bispecific PD-1a / PD1b antagonist PD-1 antibodies specific for two different epitopes) and / or PD-1 to treat diseases such as cancer. And one or more tumor antigens (eg, cancer testis (CT) antigens (ie, MAGE, NY-ESO-1); melanosite differentiation antigens (ie, Melan A / MART-1, tyrosinase, gp100); mutant antigens. (Ie, MUM-1, p53, CDK-4); specificity for both overexpressed "self" antigens (ie, HER-2 / neu, p53); and / or viral antigens (ie, HPV, EBV)). It may be beneficial to obtain a binding agent having. Binders (eg, monoclonal antibodies) can generally be made as described above. The specificity of such a binder may be rebound to a single binder using techniques widely available to those of skill in the art. In some embodiments, a plurality of monospecificity binders may be used in combination (eg, administered) to provide reagents with effective plurality of specificities.

In some embodiments, the function of a cell population expressing PD-1 (and / or another antigen in the case of a binder having multiple specificities) is targeted and inhibited, and / or the cell population thereof. The binding agents described herein may be conjugated to an active agent to eliminate. For example, a population of CD4 ⁺ T cells containing replicable HIV can be targeted and eliminated using a binder / drug conjugate (eg, an antibody-drug conjugate (ADC)). Single and / or bispecific candidate binders may be conjugated to one or more types of drugs (eg, drugs that damage DNA, drugs that target microtubules). Binders and / or derivatives thereof described herein may also be adjacent and / or conjugated to a functional agent for use in vitro and / or in vivo. For example, the binding agent may be a functional ingredient such as a cytotoxic agent or toxin and / or such as diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin. The active fragments may be adjacent and / or conjugated. Suitable functional ingredients may include radiochemicals. Binders such as antibodies can be adjacent and / or conjugated to one or more functional agents using standard techniques in the art.

In some embodiments, the binder can be administered with other agents such as, for example, anti-infective agents (eg, antibiotics, antiviral agents). For example, the binding agents described herein include monoclonal antibodies and / or other reagents such as nivolumab (MDX-1106, BMS-936558 (Topalian, et al. N. Eng. J. Med. 2012; 366). 26): 2443-2454), MDX-1106, also known as ONO-4538, a fully human IgG4 monoclonal antibody available from Bristol-Myers Squibb), Pembrolizumab (also known as MK-3475 and SCH 900475). (Humanized IgG4 monoclonal antibody available from Merck), Pidilizumab (humanized IgG1 monoclonal antibody available from CureTech), AMP-224 (B7-DC / IgG1 fusion protein available from GlaxoSmithKline / Amplimne), and / Alternatively, US Pat. No. 8,354,509B2 (Carven, et al.), US Pat. No. 8,008,449 (Korman, et al.), International Publication No. 2012/135408A1 (Manoji, et). Al.), US Patent Application Publication No. 2010/0266617 (Carven, et al.), International Publication No. 2011/11621A1 (Tyson, et al.), US Patent No. 7,488,802B2 (Al.). Collins, et al.), International Publication No. 2010/029435A1 (Simon, et al.), International Publication No. 2010/08411A2 (Olive, D.), International Publication No. 2012/1454943A1 (Langermann, et al.) , International Publication No. 2013/0435569A1 (Rolland, et al.), International Publication No. 2011/159877A2 (Kuchro, et al.), US Pat. No. 7,563,869B2 (Ono Palm.), US Patent. 7,858,746B2 (Honjo, et al.), US Pat. No. 8,728,474B2 (Onno Palm.), And / or US Pat. No. 9,067,999 (Ono). It can be combined with any of the antibodies described in (Pharm.)) Or other reagents or methods, each of which is incorporated herein by reference in its entirety. According to a preferred embodiment, any of the PD-1 binders may be fused to other binders to form bispecific binding molecules, particularly bispecific antibodies. Such bispecific molecules can be a PD1 binding agent with another PD1 binding agent, or a checkpoint inhibitor or regulator (particularly CTLA-4, LAG3, TIM3, CD137, 4-1BB, OX40, CD27). , GITR (Glucocorticoid-Induced Tumor Necrosis Factor), CD40, KIR, IDOIL-2, IL-21 and CSF-1R (Colony Stimulating Factor 1 Receptor). Other combinations and / or bispecific binding molecules are also contemplated herein, as will be appreciated by those of skill in the art.

As mentioned above, the PD-1 binders described herein (eg, PD-1 antagonists) can be used to treat and / or prevent and / or ameliorate the symptoms of HIV-induced infections. can. As is well known in the art, HIV isolates are now classified into distinct gene subtypes. HIV-1 is known to contain at least 10 subtypes (A1, A2, A3, A4, B, C, D, E, F1, F2, G, H, J and K) (Taylor et. al, NEJM, 359 (18): 1965-1966 (2008)). HIV-2 is known to contain at least 5 subtypes (A, B, C, D and E). Subtype B has been associated with HIV epidemics in homosexual and IV drug users worldwide. Most HIV-1 immunogens, laboratory adaptive isolates, reagents and mapped epitopes belong to subtype B. In sub-Saharan Africa, India and China, where the incidence of new HIV infections is high, HIV-1 subtype B accounts for only a small percentage of infections, with subtype HIV-1C being the most common. It seems to be an infection subtype. Any of these types of isolates could be addressed using the binders described herein. One or more binding agents used to prevent, treat and / or ameliorate HIV, such as, for example, protease inhibitors, HIV invasion inhibitors, reverse transcriptase inhibitors and / or antiretroviral nucleoside analogs. It may be administered with or with one or more agents capable. Suitable compounds include, for example, Agenerase (Amplenavir), Comvivir (Retrovir / Epivir), Clixivan (Indinavir), Emtriva (Emrivudine), Epivir (3tc / lamivudine), Efavirenz (3tc / lamivudine), Efavirenz (Lamivudine), Efavirenz (Lamivudine), Efavirenz (Lamivudine) ), Hivid (ddc / lamivudine), Kaletra (ropinavir), Lexiva (phosamprenavir), Norvir (ritonavir), Rescriptor (deraviudine), Retrovir / AZT (zidovudine), Reyatax (atazanavir, B) Efavirenz), Trizivir (Abacavir / Zidovudine / Lamivudine), Truvada (Emtricitabine / Tenohovir DF), Videx (ddI / Zidovudine), Videx EC (ddI, Zidovudine), Viracept (Nevirabin) Examples include Zerit (d4T / stubzine) and Ziagen (abacavir). Other suitable agents are known to those of skill in the art and may be suitable for the applications described herein. Such agents may be used either before, during or after the administration and / or use of the binding agents described herein.

As mentioned above, the PD-1 binders described herein (eg, PD-1 antagonists) can be used to treat and / or prevent and / or ameliorate the symptoms of cancer. Exemplary cancers include, for example, breast cancer, blood cancer, colon cancer, stomach cancer, colon cancer, skeletal tissue cancer, skin cancer (eg, melanoma), brain cancer, lung cancer, bladder cancer, kidney. Hmm, ovarian cancer and / or liver cancer and the like. Furthermore, the PD-1 binding agents described herein are preferably antibodies that are non-blocking to PD-1 / PD-L1 interactions, and are not particularly limited, but 135C12, 139D6, 136B4 and These, referred to as binding class 2, such as 135D1, alone or in combination with each other and / or other PD-1 antibodies, are various types of malignant tumors, especially melanoma (eg, melanoma). , Metastatic malignant melanoma), kidney cancer (eg, clear cell cancer), bladder cancer, prostate cancer (eg, cast-resistant prostate cancer), pancreatic cancer, breast cancer, colon cancer, lung cancer (eg, castor-resistant prostate cancer) For example, non-small cell lung cancer), esophageal cancer, squamous epithelial cancer of the head and neck, Mercel cell cancer, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma , Leukemia, lymphoma, sarcoma, and other neoplastic malignancies. The preferred PD-1 binding agents described above are, for example, Hodgkin's disease, such as follicular lymphoma, non-Hodgkin's lymphoma such as diffuse large B-cell lymphoma, multiple myeloid leukemia (MM), acute myeloid leukemia (AML), acute. Particular focus has been placed on the treatment of hematological malignancies such as lymphocytic leukemia (ALL) and myeloid dysplasia syndrome. The binders described therein may also be used to treat other types of cancer, as will be appreciated by those of skill in the art.

In some embodiments, the one or more PD-1 binding agents are, for example, alkylating agents (eg, any nitrogen mustard, nitrosolea, tetrazine, aziridine, cisplatin and / or derivatives thereof), metabolism. Antagonists (eg, methotrexate, pemetrexed, fluoropyrimidin and / or derivatives thereof), antimicrotubes (eg, bincaalkyloid, taxan, podophyllotoxin and / or derivatives thereof), topoisomerase I and / or II Inhibitors (eg, camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxanthrone, teniposide, novobiocin, mervalon, acralvisin and / or derivatives thereof) and / or cytotoxic antibiotics (eg, any anthracycline, actino). May be combined with one or more agents used to prevent, treat and / or ameliorate cancer, such as mycin, bleomycin, plicamycin and mitomycin and / or derivatives thereof), and / or with them. It may be administered together or together. Further or instead, one or more binders to treat, prevent and / or ameliorate cancer, such as, for example, nivolumab, pembrolizumab, pigrizumab and / or other similar agents and / or derivatives thereof. However, it may be combined with one or more other binders available to those skilled in the art. One or more PD-1 binders may be used alone or in combination with other binders that target PD-1, PDL-1 and / or other immune checkpoint effectors. These are pulsed with other antitumor or immunogenic agents (eg, attenuated cancer cells, tumor antigens (such as recombinant proteins, peptides, and sugar molecules), eg tumor-derived antigens or nucleic acids). Trans in genes encoding antigen-presenting cells such as, immunostimulatory cytokines (eg, IL-2, IFNa2, GM-CSF), and / or immunostimulatory cytokines (eg, but not limited to, GM-CSF, etc.). Ffected cells); standard cancer treatment (eg, chemotherapy, radiotherapy, or surgery); or in combination with VEGF, EGFR, Her2 / neu, VEGF receptors, other drugs against other growth factor receptors; May be used. According to a preferred embodiment, one or more PD-1 binding agents can be used as a vaccine and / or, in particular, CTLA-4, LAG3, TIM3, CD137, 4-1BB, OX40, CD27, GITR (glucocorticoid-inducible). Tumor necrosis factor), CD40, KIR, IDOIL-2, IL-21 and / or immune checkpoint regulators acting on CSF-1R (colony stimulating factor 1 receptor) in combination with therapeutic compositions and / or sequential treatment. Form a kit for administration. Other suitable agents are known to those of skill in the art and may be suitable for the applications described herein. Such agents can be used either before, during or after the administration and / or use of the binding agents described herein.

As mentioned above, the PD-1 binders described herein (eg, PD-1 agonists) can be used to treat and / or prevent and / or ameliorate the symptoms of autoimmunity. Typical autoimmune conditions involve PD-1 maintaining self-tolerance, such as systemic lupus erythematosus (SLE), type I diabetes, rheumatoid arthritis, glomerulonephritis, and polysclerosis. And / or any condition with inflammatory T cells (eg, autoreactive or self-antigen-specific T cells) can be included. Such PD-1 binders may be combined with other agents such as, for example, anti-CTLA-4 agents (eg, ipilimumab). One or more binding agents can be used, for example, glucocorticoids, cell growth inhibitors (eg, alkylating agents, anti-metabolism agents, methotrexate, azathioprine, mercaptopurine, cytotoxic antibiotics (eg, dactinomycin, anthra). Drugs that act on cyclins, mitomycin C, bleomycin, mitramycin), antibodies (eg, Atgam, Thermoglobuline, Simulect, Zenapax), immunophilins (eg, cyclosporine, tacrolimus, silolimus), interferons, opioids, TNF binding agents, eg. , Remicade, Enbrel, Humira), mycophenorate, fingerolimod, myriocin and / or derivatives thereof, eg, in combination with one or more agents that can be used to prevent, treat and / or ameliorate autoimmunity, and / Or may be administered together or together. Other suitable agents are known to those of skill in the art and may be suitable for the uses described herein. Such agents are described herein. Can be used either before, during or after the administration of the binding agent and / or the use of the method.

In some embodiments, the binder may be adjacent and / or conjugated to one or more detectable labels. For example, as suitable detectable labels, for example, fluorosane (eg, Dlyght, Cy3, Cy5, FITC, HiLyte Fluor 555, HiLyte Fluor 647; 5-carboxy-2,7-dichlorofluorescein; 5-carboxyfluoresane (5). -FAM); 5-HAT (hydroxytryptamine); 5-hydroxytryptamine (HAT); 6-JOE; 6-carboxyfluorescein (6-FAM); FITC; 6-carboxy-1,4-dichloro-2', 7 '-Dichlorofluorescein (TET); 6-carboxy-1,4-dichloro-2', 4', 5', 7'-tetrachlorofluorescein (HEX); 6-carboxy-4', 5'-dichloro-2 ', 7'-Dimethoxyfluolecein (JOE); Alexa fluoro (eg, 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 680, 700, 750); BODIPY phosphors (eg, 492/515, 493/503, 500/510, 505/515, 530/550, 542/563, 558/568, 564/570, 576/589, 581/591). , 630 / 650-X, 650 / 665-X, 665/676, FL, FL ATP, FI-Ceramide, R6G SE, TMR, TMR-X Conjugate, TMR-X, SE, TR, TR ATP, TR- X SE)), Rhodamine (eg 110, 123, B, B 200, BB, BG, B extra, 5-carboxytetramethylrhodamine (5-TAMRA), 5 GLD, 6-carboxyrhodamine 6G, lysamine, lysamine Rhodamine B, Faricidine, Faroidin, Red, Rhod-2, ROX (6-carboxy-X-Rhodamine), 5-ROX (Carboxy-X-Rhodamine), Sulforodamine B can C, Sulforodamine G Extra, TAMRA (6- Carboxytetramethylrhodamine), tetramethylrhodamine (TRITC), WT), Texas Red and / or Texas Red-X and the like. Other detectable labels known in the art may also be suitable for use. Binders, such as antibodies, can be adjacent and / or conjugated to one or more detectable labels using standard techniques in the art.

In certain embodiments, the nucleic acid molecule encoding one or more binders described herein may be inserted into one or more expression vectors as discussed in more detail below. In such embodiments, the binder may be encoded by the nucleotide corresponding to the amino acid sequence. Certain combinations of nucleotides (codons) encoding various amino acids (AAs) are well known in the art as described in the various references used by those of skill in the art (eg, Lewin, et al. B. Genes V, Oxford University Press, 1994). The nucleotide sequence encoding the amino acid of the binder can be confirmed, for example, by referring to Table 9. Nucleic acid variants may use any combination of nucleotides encoding the binder.

Those skilled in the art can understand that the nucleotide sequence encoding a particular amino acid sequence can be easily derived from the amino acid sequences and information shown in Table 9 (and, in some embodiments, Tables 5-8). For example, from the amino acid sequence DDFLH (SEQ ID NO: 1) and the information shown in Table 9, it can be inferred that this amino acid sequence can be encoded by the nucleotide sequence GAT GAT TTT TTA CAT (SEQ ID NO: 191). Those skilled in the art can deduce the nucleotide sequence encoding any of SEQ ID NOs: 2 to 190 in the same manner and understand that such a nucleotide sequence is contemplated herein. In addition, when the binding agent is an antibody, a nucleotide sequence encoding a variable region thereof is cloned into an expression vector to express a predetermined preparation (for example, a humanized antibody). And / or can be isolated from hybridoma cells. An exemplary nucleic acid sequence encoding the variable region sequences of SEQ ID NOs: 139-190 is shown in Table 10.

As will be appreciated by those of skill in the art, the sequences used above may be modified for use, for example by codon optimization or other techniques available in the art. Methods of making such preparations are described herein and / or are well known and available to those of skill in the art.

Hybridoma cells derived from mice immunized with the PD-1 antigen / immunogen to identify the amino acid sequence of the variable region of interest (eg, CDR) are subjected to the functional assays described herein and those of skill in the art. Select using readily available cloning techniques. For example, to isolate and sequence nucleic acids encoding heavy and light chain variable regions of selected hybridomas, total RNA is extracted from fresh hybridoma cells using the TRIzol reagent according to the manufacturer's protocol. .. The cDNA can be synthesized from RNA using standard techniques using isotype-specific antisense or universal primers (eg, according to the technical manual of the PrimeScript ™ 1st Strand cDNA Synthesis Kit). A polymerase chain reaction (PCR) can then be performed to amplify the nucleic acid encoding the (heavy and light chain) variable regions of the antibody produced by the selected hybridoma, which is then standard cloning vector. Can be cloned and sequenced separately within. Colony PCR screening can then be performed to identify clones with the correct size insert. Preferably, at least 5 single colonies with the correct size inserts are sequenced for each antibody variable region. Standard protocols can then be used for expression and purification of anti-PD-1 antibodies. For example, hybridoma clones may be grown in serum-free medium, cell culture broths centrifuged and further filtered. The filtered supernatant containing the antibody may then be loaded onto an affinity column (eg, a protein A column), washed and then eluted with a suitable buffer (eg, Pierce IgG elution buffer). The elution fraction may be pooled and buffer exchanged in PBS (pH 7.2). Purified antibodies can then be analyzed by SDS-PAGE and Western blotting using standard protocols for molecular weight, yield and purity. Next, a suitable column for biophysical property analysis (eg, TSK GEL-G3000 SWXL column (Tosoh)) to ensure high antibody purity (usually> 90%) with low protein aggregate presence. )), Size exclusion chromatography HPLC may be performed. These techniques were used to isolate and sequence nucleic acids encoding SEQ ID NOs: 1-190 from selected cells and other sources. These techniques, variations thereof and / or other techniques may also be used for these purposes as will be appreciated by those skilled in the art.

Nucleic acid molecules encoding one or more PD-1 binders may be contained within viral and / or non-viral vectors. In one embodiment, a DNA vector is utilized to deliver a nucleic acid encoding one or more PD-1 binders to a patient. In doing so, various strategies can be used to improve the efficiency of such mechanisms, eg, self-replicating virus replicons (Caley, et al. 1999. Vaccine, 17: 3124-2135; Dubensky, et al. 2000. Mol. Med. 6: 723-732; Leitner, et al. 2000. Cancer Res. 60: 51-55), Codon Optimization (Liu, et al. 2000. Mol. Ther., 1: 497-500). Dubensky, supra; Hung, et al. 2001.J. Virus. 75: 4947-4951), in vivo electroporation (Widera, et al. 2000. J. Immunol. 164: 4635-3640), costimulatory. Integration of nucleic acid molecules encoding molecules, cytokines and / or chemokines (Xiang, et al. 1995.Immunitity, 2: 129-135; Kim, et al. 1998. Eur. J. Immunol., 28: 1089-1103; Iwasaki, et al. 1997.J. Immunol. 158: 4591-3601; Sheerlink, et al. 2001. ), Sequences for targeting endocytosis or ubiquitin processing pathways (Thomson, et al. 1998. J. Virus. 72: 2246-2252; Velders, et al. 2001. J. Immunol. 166: 5366-5373). , Prime-boost regimen (Guruntan, supra; Sullivan, et al. 2000. Nature, 408: 605-609; Hanke, et al. 1998. Vaccine, 16: 439-445; Amara, et al. 2001. Science, 292. : 69-74), use of proteasome-sensitive cleavage sites, and use of mucosal delivery vectors such as, for example, Salmonella (Darji, et al. 1997. Cell, 91: 765-775; Woo, et al. 2001. Vaccine). , 19: 2945-2954) and the like. Other methods are known in the art and some of them are described below. Various viral vectors that have been successfully utilized to introduce nucleic acids into the host include, for example, retroviruses, adenoviruses, adeno-associated viruses (AAV), herpesviruses and poxviruses. Vectors can be constructed using standard recombinant techniques widely available to those of skill in the art. Such techniques include, for example, Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technique, Entry Technology, It can be found in general molecular biology references such as Press, San Diego, CA) and PCR Protocols: A Guide to Techniques and Applications (Innis, et al. 1990. Academic Press, San Diego, ca). .. A "non-viral" plasmid vector may also be suitable in certain embodiments. Preferred plasmid vectors may be compatible with bacterial, insect and / or mammalian host cells. Such vectors include, for example, PCR-ii, PCR3 and pcDNA3.1 (Invitrogen, San Diego, CA), pBSii (Stratagene, La Jolla, CA), pet15 (Novagen, Madison, WI), pGEX (Pharmacia). Piscataway, NJ), pEGFp-n2 (Clontech, Palo Alto, CA), pETl (Bluebacii, Invitrogen), pDSR-α (International Publication No. 90/14363) and pFASTBACdual (Gibco-BRL, Grand) (Registered Trademarks) plasmid derivatives (high copy count COLe1-based phagemid, Stratage Cloning Systems, La Jolla, CA), PCR cloning plasmids designed for cloning TAQ-amplified PCR products (eg, TOPO ™ TA cloning (eg, TOPO ™ TA cloning). Kits, PCR2.1® plasmid derivatives, Invitrogen, Carlsbad, CA) and the like. Bacterial vectors can also be used. Such vectors include, for example, Shigella, Salmonella, Vibrio cholerae, Lactobacillus, Shigella Calmette Guerin (BCg), and Bacillus Calmette Guerin (BCG). , International Publication No. 88/6626; International Publication No. 90/0594; International Publication No. 91/13157; International Publication No. 92/1796 and International Publication No. 92/21376) and the like. Numerous other non-viral plasmid expression vectors and expression systems are known in the art and can be used. Other delivery techniques such as DNA-ligand complex, adenovirus-ligand-DNA complex, direct injection of DNA, CaPO ₄ precipitation, gene gun technology, electroporation and colloidal dispersion systems are also acceptable. Colloidal dispersion systems include lipid-based systems including polymer complexes, nanocapsules, microspheres, beads and oil emulsions in water, micelles, mixed micelles and liposomes. A preferred colloidal system is a liposome, which is an artificial membrane vesicle useful as a delivery vehicle in vitro and in vivo. RNA, DNA and intact virions can be encapsulated inside the aqueous solution and delivered to cells in bioactive form (Frayy, R., et al., 1981, Trends Biochem. Sci., 6:77). The composition of the liposomes is usually a combination of phospholipids, especially high phase transition temperature phospholipids, in combination with steroids, especially cholesterol. Other phospholipids or other lipids can also be used. The physical properties of liposomes depend on pH, ionic strength and the presence of divalent cations. Examples of lipids useful in liposome fabrication include phosphatidyl compounds such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides and gangliosides. Particularly useful are diacylphosphatidylglycerols in which the lipid moiety has 14-18 carbon atoms, in particular 16-18 carbon atoms and is saturated. Exemplary phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

Cultured cells containing the vector are also provided. A cultured cell is a vector-transfected cultured cell or a progeny of that cell, which expresses an immunogenic polypeptide. Suitable cell lines are known to those of skill in the art and are commercially available, for example, through the American Type Culture Collection (ATCC). Transfected cells can be used in methods of producing immunogenic polypeptides. The method comprises culturing cells containing the vector under conditions that allow the expression of the immunogenic polypeptide, optionally under the control of the expression sequence. Immunogenic polypeptides can be isolated from cells or culture medium using standard protein purification methods.

One of ordinary skill in the art has a number of suitable techniques for identifying biological samples containing proteins that bind to the binders (eg, antibodies) described herein. For example, immunoprecipitation or other capture-type assays can be used to utilize the antibody to isolate PD-1. This well-known technique is performed by attaching an antibody to a solid support or chromatographic material (eg, protein A, protein G and / or protein L). The bound antibody is then introduced into a solution containing or believed to contain PD-1 (eg, HIV-infected T cell lysates). Thereby, PD-1 binds to the antibody and washes away the unbound substance under the condition that PD-1 remains bound to the antibody. The bound protein is then separated from the antibody and the desired analysis is performed. Similar methods of isolating proteins using antibodies are well known in the art. Binders (eg, antibodies) can also be used to detect PD-1 in biological samples. Antibodies can be used, for example, in assays such as flow cytometric analysis, ELISA, immunoblotting (eg, western blot), insitu detection, immunocytochemistry and / or immunohistochemistry. Methods of performing such assays are well known in the art.

The binders described herein can also be used to identify the presence of a disease state in a patient, predict prognosis, or identify the effectiveness of a chemotherapeutic agent or other treatment regimen. Expression profile assays, as described herein or as known in the art, can be used to identify relative levels of expression of PD-1. The expression level is then correlated with a reference (eg, control) level to determine if a particular disease is present in the patient, the patient's prognosis, or whether a particular treatment regimen is effective. can do. For example, if a patient is being treated with a particular anti-infectious disease treatment regimen, an increase or decrease in PD-1 expression levels in the patient's tissue (eg, peripheral blood, breast tissue biopsy) will be hosted by that treatment regimen. It can be expressed as exacerbating or ameliorating the amount of infectious agent within. An increase or decrease in expression can indicate that the treatment regimen has or does not have the desired effect, and thus another treatment can be selected.

In addition, the binders described herein can also be used, for example, as reagents in drug screening assays for testing new drug candidates. The reagent can be used to confirm the effect of a candidate drug on the expression of an immunogenic target in a cell line or in a patient's cell or tissue. Expression profiling techniques may be combined with high-throughput screening techniques (eg, Zlokarnik, et al., Science) to allow rapid identification of useful compounds and to monitor the effectiveness of treatment with candidate agents. 279, 84-8 (1998)). Candidate agents may be chemical compounds, nucleic acids, proteins, antibodies, or derivatives thereof, and may be either natural or synthetically derived. Candidate agents identified in this way can be utilized, for example, as pharmaceutical compositions for administration to patients or for use in other screening assays.

In some embodiments, the binder is in purified form. A "purified" binder (eg, an antibody) is at least about 50% of the protein and / or other components initially included together (eg, in the case of a monoclonal antibody, as part of a hybridoma supernatant or ascites preparation). It may be separated from. The purification binder (eg, antibody) may be isolated from at least about 50%, 60%, 75%, 90% or 95% of the protein and / or other components initially included together.

The polypeptides and nucleic acids described herein may be combined with one or more pharmaceutically acceptable carriers prior to administration to the host. A pharmaceutically acceptable carrier is not a biologically or otherwise undesired substance, eg, the substance does not cause any undesired biological effects, or is a pharmaceutical composition comprising it. It can be administered to a subject without harmful interaction with any of the other components. The carrier will necessarily be selected to minimize the degradation of the active ingredient and to minimize the adverse side effects in the subject, as is well known to those of skill in the art. Suitable pharmaceutical carriers and their preparations are described, for example, in Remington's: The Science and ^Concoction of Pharmacy, 21st Edition, David B. et al. Troy, ed. , Lippicott Williams & Wilkins (2005). Typically, an appropriate amount of pharmaceutically acceptable salt is used in the preparation to make the preparation isotonic. Examples of pharmaceutically acceptable carriers include, but are not limited to, buffers such as sterile water, saline, Ringer's and dextrose solutions. The pH of the solution is generally about 5-8 or about 7-about 7.5. Other carriers include sustained release formulations such as, for example, a semi-permeable matrix of solid hydrophobic polymers containing polypeptides or fragments thereof. The matrix may be in the form of an article, such as a film, liposome or microparticle. It will be apparent to those skilled in the art that a given carrier may be more preferred, for example, depending on the route of administration and the concentration of the composition being administered. The carrier is suitable for administering a polypeptide and / or a fragment thereof to a human or other subject. In addition to the immunogenic polypeptide, the pharmaceutical composition may also include carriers, thickeners, diluents, preservatives, surfactants, adjuvants, immunostimulators. The pharmaceutical composition may also contain one or more active ingredients such as antibacterial agents, anti-inflammatory agents and anesthetics. The pharmaceutical composition is taken orally, parenterally, by inhalation spray, transrectally, intranodule or topically in a unit dose preparation containing conventional pharmaceutically acceptable carriers, adjuvants and vehicles. Can be administered. As used herein, the term "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" is one or more suitable for achieving or enhancing delivery of a nucleic acid, polypeptide or peptide as a pharmaceutical composition. Means the preparation material of. A "pharmaceutical composition" is a composition comprising a therapeutically effective amount of a nucleic acid or polypeptide. The terms "effective amount" and "therapeutic effective amount" mean the amount of binder, nucleic acid, etc. used to observe the desired therapeutic effect (eg, restore T cell function), respectively.

One or more disease states (and / or derivatives thereof) in a mammalian host comprising administering to the mammal one or more binding agents (and / or derivatives thereof) described herein in at least one effective dose. For example, methods of treating HIV or cancer) are also provided. In some embodiments, the binder is a monoclonal antibody, or fragment or derivative thereof, comprising one or more of SEQ ID NOs: 1-190 and / or Tables 1A and 1B. For example, in certain preferred embodiments, the binder, or at least one of the binders in the combination, is SEQ ID NOs: 143 and 155 (eg, antibody A35795), SEQ ID NOs: 166 and 180 (eg, antibody A35774). ), SEQ ID NOs: 176 and 189 (eg, such as antibody 135C H1cL1b), and / or SEQ ID NOs: 176 and 190 (eg, such as antibody 135C H1cL1c), and / or conservative or non-conservative substitution variants thereof. You can go out. Preferably, the CDR regions of such binders (ie, SEQ ID NOs: 1-138) have not been substituted. The one or more binders may be about 0.1 to about 50 mg / kg, about 1 to about 30 mg / kg, or about 5 to about 30 mg / kg (eg, about 0.1, 0.2, 0. 25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35 or 40 mg / kg) It can be administered in doses. The amount of each of the individual binders may be the same or different. In certain embodiments, the one or more binders can be administered to the mammal once or multiple times at about 10 mg / kg (eg, intradermally, intravenously, orally, transrectally). If administered multiple times, the dose may include approximately the same or different amounts of binder in each dose. Administrations may be separated in time at the same or different intervals. For example, administration is about 6, 12, 24, 36, 48, 60, 72, 84 or 96 hours, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1.5 years, 2 years, 3 years, 4 years, 5 years, or It can be separated by the period before, after and / or between any of these periods. In some embodiments, the binder can be administered, for example, with other agents (eg, anti-infective agents and / or chemotherapeutic agents). Such other agents can be administered approximately simultaneously with the binder or at different time points and / or frequencies. Other embodiments of such methods may also be appropriate for those skilled in the art to readily identify.

To assist one of ordinary skill in the art in using the binders described herein (eg, antibodies, etc.), it may be provided in the form of a kit. Kits are provided that include such a binder (eg, an antibody, etc.) and optionally other components necessary to detect cells expressing PD-1 using that antibody. The kit binder (eg, antibody, etc.) may be provided in any suitable form such as freezing, lyophilization, or in a pharmaceutically acceptable buffer such as TBS or PBS. The kit includes, for example, a buffer (eg, TBS, PBS), a blocking agent (a solution containing defatted milk powder, normal serum, Tween-20 detergent, BSA, or casein), and / or a detection reagent (eg, goat anti-mouse). In, such as IgG biotin, streptavidin-HRP conjugate, allophicocyanin, B-phycoerythrin, R-phycoerythrin, peroxidase, detectable labels and other labels and / or staining kits (eg, ABC staining kits, Pierce)). Other reagents required to utilize the antibody in vitro or in vitro may also be included. The kit is for using the antibody in the commonly used assays described above, such as flow cytometry analysis, ELISA, immunoblotting (eg Western blot), insitu detection, immunocytochemistry, immunohistochemistry, etc. Other reagents and / or instruction manuals may also be included. In one embodiment, the kit provides the binder in purified form. In yet another embodiment, the binder may be provided in biotinylated form, alone or with an avidin-conjugated detection reagent (eg, an antibody). In yet another embodiment, the kit comprises a binder having one or more detectable labels that can be used to directly detect PD-1. The buffers and the like required to use any of these systems are well known in the art and / or may be prepared by the end user or provided as a component of the kit. The kit may also include a solid support containing positive and negative control proteins and / or tissue samples. For example, a kit for performing a spotting or western blot type assay may be a control cell or tissue lysate for use in SDS-PAGE, or a pre-fixed control sample with additional space for the experimental sample. May include a nylon membrane or other membrane containing. Kits for visualizing PD-1 in cells on slides may include formatted slides containing control cells or tissue samples with additional space for experimental samples. Other embodiments of the kit are also contemplated herein, as will be appreciated by those of skill in the art.

Therefore, the present disclosure provides a binder that binds to PD-1 operably or antagonistically. In some embodiments, the binder is a polypeptide selected from the group consisting of SEQ ID NOs: 1-190 and / or comprising at least one amino acid sequence shown in Tables 1A and 1B. In some embodiments, the binder is a polypeptide comprising one or more combinations of SEQ ID NOs: 1-138 and / or SEQ ID NOs: 139-190 (eg, as shown in Tables 1A and / or 1B). be. In some embodiments, the binder is an antibody. In some embodiments, the binder is a polypeptide, such as an antibody, comprising a heavy chain CDR1 amino acid sequence selected from the group consisting of SEQ ID NOs: 1-23. In some embodiments, the binder is a polypeptide, such as an antibody, comprising a heavy chain CDR2 amino acid sequence selected from the group consisting of SEQ ID NOs: 24-46. In some embodiments, the binder is a polypeptide, such as an antibody, comprising a heavy chain CDR3 amino acid sequence selected from the group consisting of SEQ ID NOs: 47-69. In some embodiments, the binder is a polypeptide, such as an antibody, comprising a light chain CDR1 amino acid sequence selected from the group consisting of SEQ ID NOs: 70-92. In some embodiments, the binder is a polypeptide, such as an antibody, comprising a heavy chain CDR2 amino acid sequence selected from the group consisting of SEQ ID NOs: 93-115. In some embodiments, the binder is a polypeptide, such as an antibody, comprising a heavy chain CDR3 amino acid sequence selected from the group consisting of SEQ ID NOs: 116-138. In certain embodiments, the binder is a polypeptide, such as an antibody, comprising one or more of SEQ ID NOs: 164-190 and / or a conservative or non-conservative substitution derivative thereof. In certain embodiments, the binder is a polypeptide, such as an antibody, comprising one or more of SEQ ID NOs: 139-150 and one or more of SEQ ID NOs: 151-163. In certain embodiments, the binder is a polypeptide, such as an antibody, comprising one or more of SEQ ID NOs: 164 to 177 and one or more of SEQ ID NOs: 178 to 190. For example, in certain preferred embodiments, at least one of the binding agents or binding agents in the combination is SEQ ID NOs: 143 and 155 (eg, such as antibody A35795), SEQ ID NOs: 166 and 180 (eg, such as antibody A35774). SEQ ID NOs: 176 and 189 (eg, antibody 135C H1cL1b) and / or SEQ ID NOs: 176 and 190 (eg, antibody 135C H1cL1c), and / or conservative or non-conservative substitution derivatives thereof (eg, Table 5). It may be an antibody containing (as shown in 8). In certain embodiments, the binder comprises a combination of CDRs shown in Tables 1A and / or 1B and / or has the properties described in any one or more of Tables 3 and / or Tables 11-13.

In some embodiments, the binding agent is a human antibody, human IgG1, human IgG1, human IgG2, human IgG2a, human IgG2b, human IgG3, human IgG4, human IgM, human IgA, human IgA1, human IgA2, human IgD, Human IgE, dog antibody, dog IgGA, dog IgGB, dog IgGC, dog IgGD, chicken antibody, chicken IgA, chicken IgD, chicken IgE, chicken IgG, chicken IgM, chicken IgY, goat antibody, goat IgG, mouse antibody, mouse IgG , Derived from or related to pig antibodies and / or rat antibodies and / or derivatives thereof (eg, by sequence or origin). In some embodiments, the derivative is, for example, Fab, _Fab2 , _Fab'single -stranded antibody, Fv _, single-stranded (eg, scFv), _VHH / _VH , _unispecific antibody, two. Multispecific antibody, trispecific antibody, multispecific antibody, multivalent antibody, chimeric antibody, canine-human chimeric antibody, canine-mouse chimeric antibody, antibody containing canine Fc, humanized antibody, human antibody, canineized antibody, It can be selected from the group consisting of CDR grafted antibody, shark antibody, nanobody and / or camel antibody. Such antibodies, fragments, etc. can be made using any of the widely available techniques for that purpose. For example, humanized antibodies can be used and / or widely available using the procedures described in US Pat. No. 9,090,994 (Zhang et al.) And / or systems (eg, GenScript). It can be made using a private company such as (registered trademark) (eg, one available from a FASEBA system). In certain embodiments, the binding agent is one or more of the amino acid sequences of SEQ ID NOs: 139-190 (or encoded by the nucleic acid sequence encoding such an amino acid) and / or derivatives or variants thereof (eg, eg). It may be a humanized antibody containing the conservative substitution derivative or variant). Other embodiments are also contemplated as understood by those of skill in the art.

In certain embodiments, the binder is at least one of SEQ ID NOs: 139-150 and at least one of SEQ ID NOs: 151-163 and / or a derivative or variant thereof (eg, a conservative substitution derivative thereof). Alternatively, it may be a humanized antibody containing a variant). In certain embodiments, the binding agent is shown in Table 12, humanized antibody A35796 (including SEQ ID NOs: 140 and 152), humanized antibody A35793 (including SEQ ID NOs: 141 and 153), humanized antibody A35518 (including SEQ ID NOs). 142 and 154), humanized antibody A35795 (including SEQ ID NOs: 143 and 155), humanized antibody A35797 (including SEQ ID NOs: 144 and 156), humanized antibody A35799 (including SEQ ID NOs: 145 and 157), human. Humanized antibody A35805 (including SEQ ID NOs: 146 and 158); humanized antibody 137F VH1 / VL1 (including SEQ ID NOs: 147 and 159), humanized antibody 137F VH2 / VL2 (including SEQ ID NOs: 148 and 160), humanized antibody. Shown by 137F VH1b / VL1b (including SEQ ID NOs: 149 and 161), humanized antibody 137F VH1c / VL1c (including SEQ ID NOs: 150 and 162), or humanized antibody 137F VH1b / VL1d (including SEQ ID NOs: 149 and 163). Almost the same binding affinity for human and monkey PD-1 (ka (M ^-1 / s ^-1 ), kd (s ^-1 ), and KD (M)) (ie, about the same). Within any range of 250%, about 200%, about 150%, about 100%, about 75%, about 50%, about 25%, about 20%, about 15%, or about 10%; and / Or at least 10 nM affinity ( ^KD10-8M )). In certain embodiments, the 137F VL1d may be combined (or paired) with any other 137F variable heavy chain.

In other embodiments, the binder is at least one of SEQ ID NOs: 164 to 177 and at least one of SEQ ID NOs: 178 to 190, and / or a derivative or variant thereof (eg, a conservative substitution thereof). It may be a humanized antibody containing a derivative or variant). In certain embodiments, the binding agent is shown in Table 13, humanized antibody A35775 (including SEQ ID NOs: 164 and 178), humanized antibody A35783 (including SEQ ID NOs: 165 and 179), humanized antibody A35774 (including SEQ ID NOs). 166 and 180), humanized antibody A36443 (including SEQ ID NOs: 167 and 181), humanized antibody A35777 (including SEQ ID NOs: 168 and 182), humanized antibody A35789 (including SEQ ID NOs: 169 and 18), human. Humanized antibody A36448 (including SEQ ID NOs: 170 and 184), or humanized antibody A36437 (including SEQ ID NOs: 171 and 185); humanized antibody 135C VH1 / VL1 (including SEQ ID NOs: 172 and 186), humanized antibody 135C VH2. / VL2 (including SEQ ID NOs: 173 and 187), humanized antibody 135C VH3 / VL3 (including SEQ ID NOs: 174 and 188), humanized antibody 135C VH1b / VL1b (including SEQ ID NOs: 175 and 189), humanized antibody 135C. Humans and humans with VH1c / VL1b (including SEQ ID NOs: 176 and 189), humanized antibody 135C VH1c / VL1c (including SEQ ID NOs: 176 and 190), or humanized antibody 135C VH1d / VL1c (including SEQ ID NOs: 177 and 190). Almost the same binding affinity (ie, about 25% of them) as the binding affinity for PD-1 in monkeys (ka (M ^-1 / s ^-1 ), kd (s ^-1 ), and KD ( _M )). , Is in the range of about 20%, about 15%, or about 10%; and / or exhibits an affinity of at least 10 nM ( ^{KD 10-8} _M ).

In some embodiments, the binding agent has at least first and second specificity, the first specificity is for PD-1, and the second specificity is for a different antigen (eg, HIV, etc.). Infectious agent for antigens (eg Env) and / or tumor antigens. In some embodiments, the binder and / or its derivative may comprise a detectable label fixably attached to it. In some embodiments, the binder and / or a derivative thereof is an effector moiety fixedly attached to it (eg, a cytotoxic agent, a toxin, a diphtheria A chain, an exotoxin A chain, a ricin A chain, an abrin A chain). , Curcin, crotin, phenomycin, enomycin and radiochemicals). In some embodiments, polynucleotides encoding one or more binders (eg, as expression vectors) are also provided. Host cells containing and / or expressing the polypeptide product of such polynucleotides are also provided. In some embodiments, at least one binder or derivative; at least one isolated polynucleotide; at least one expression vector; and / or at least one host cell; or a combination thereof; and a pharmaceutically acceptable carrier. Also provided are compositions comprising.

The present disclosure further relates to a method of detecting PD-1 on cells, the step of contacting a test biological sample with a binder or derivative described herein, and the biological sample or component thereof. Also provided is a method comprising a step of detecting a binder bound to. Such a method may be an in vivo method or an in vitro method. In some embodiments, the method comprises comparing the amount bound to the test biological sample or its constituents to the amount bound to the control biological sample or its constituents, comprising the step of comparing the control biological sample or its constituents. An increase in the amount bound to the test biological sample or component thereof compared to the amount bound to the component indicates the presence of cells expressing PD-1 in the test biological sample (eg, mammalian blood). In some embodiments, the candidate binder is assayed by the Functional Exhaustion Recovery Assay (EFRA); the affinity of the candidate binder for PD-1 is identified; and the nucleotide sequence of the CDR of the candidate binder is identified. Provides a system for identifying PD-1 antibody binders.

In some embodiments, a kit for detecting intracellular or intracellular expression of PD-1 and comprising a binder or a derivative thereof and instructions for use is provided. In some embodiments, the binder and / or its derivative is in a lyophilized form.

In some embodiments, the present disclosure is a method of treating, preventing and / or ameliorating an infectious disease, cancer and / or autoimmunity in a mammal, comprising a binding agent or a derivative thereof. Provided are methods comprising administering to a mammal at least one effective dose of. In some embodiments, the infectious disease is the human immunodeficiency virus (HIV). In some embodiments, the binder and / or derivative thereof used to treat an infectious disease and / or cancer is a PD-1 antagonist. In some embodiments, the binder and / or derivative thereof used to treat an autoimmune condition is a PD-1 agonist. In some embodiments, multiple doses are administered to the animal. In some embodiments, the binder and / or a derivative thereof is about 0.1 to about 50 mg / kg, about 1 to about 30 mg / kg, or about 5 to about 30 mg / kg (eg, about 0.1, About 0.2, about 0.25, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 2 , About 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 18, about A dose of 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 35, or about 40 mg / kg). Can be administered at.

The present disclosure also provides a combination of PD-1 binders. In some embodiments, the combination comprises a first binder that blocks the interaction of PD-1 with PD-L1 and a second binder that does not block the interaction of PD-1 with PD-L1. .. As an exemplary combination, for example, one or more of the blocking anti-PD-1 antibodies and, for example, 135C12 (SEQ ID NOs: 17, 40, 63, 86, 109, and 132), 139D6 (SEQ ID NOs: 2, 25, 48). , 71, 94, and 117), 135D1 (SEQ ID NOs: 3, 26, 49, 72, 95, and 118), and / or 136B4 (SEQ ID NOs: 19, 42, 65, 88, 111, and 134). It may include one or more of the non-blocking binders described herein, such as any one or more of those comprising: Exemplary embodiments include a blocking antibody MK3475 (pembrolizumab) or a blocking binder comprising the amino acid sequences of SEQ ID NOs: 8, 31, 54, 77, 100, and 123 (137F2) and SEQ ID NOs: 17, 40, 63, Includes a combination with a non-blocking PD-1 binding agent comprising the amino acid sequences 86, 109, and 132 (135C12). As shown in FIGS. 9A and 9B, both of these exemplary combinations enhance the relief of functional exhaustion of HIV-specific CD8 ⁺ T cells more than achieved with either antibody alone. And enhance its proliferation. Other exemplary non-blocking antibodies that may be useful in combination with the blocking antibody MK3475 or 137F2 are non-blocking P2 patch-specific (M4 (FIGS. 3A, B)) binder 135D1 (SEQ ID NOs: 3, 26, 49, 72, 95 and 118) and / or binder 139D6 (including SEQ ID NOs: 2, 25, 48, 71, 94, and 117), and / or non-blocking P2 patch-specific (M17 (FIGS. 3A, E)). ) Binder 136B4 (including SEQ ID NOs: 19, 42, 65, 88, 111, and 134). Other combinations may be appropriate to be identified by those of skill in the art.

Such combinations can be used for any use described herein or as confirmed by one of ordinary skill in the art. For example, such combinations may be used in the methods described herein to treat, prevent and / or ameliorate infectious diseases, cancer and / or autoimmunity in mammals. In certain embodiments, a method of treating inflammation in a subject comprises administering a combination of at least two antagonists PD-1 binding agents, each of which is specific to a different epitope on PD-1. At least one of the binders is blocking the interaction between PD-1 and PD-L1, and at least one of the binders is the interaction between PD-1 and PD-L1. It also provides a non-blocking method. The present disclosure is also a method of treating a chronic neurodegenerative condition in a subject, comprising administering a combination of at least two antagonists PD-1 binding agents, each of which is a different epitope on PD-1. At least one of the binders is blocking the interaction of PD-1 and PD-L1, and at least one of the binders is with PD-1 and PD-L1. It also provides a method that is non-blocking to the interaction. In certain embodiments, the chronic neurodegenerative condition is Alzheimer's disease. The present disclosure is also a method of inhibiting the growth of tumor cells in vivo, eg, human tumor cells, such as in the treatment of cancer, wherein a combination of at least two antagonists PD-1 binding agents is administered. Each of the binding agents has specificity for different epitopes on PD-1, and at least the first binding agent of the binding agent is blocking property for the interaction between PD-1 and PD-L1. , And a method in which at least the second binding agent of the binding agent is non-blocking to the interaction between PD-1 and PD-L1 is also provided. In certain embodiments, the combinations include, for example, RASKGVSTSGYSYLH (SEQ ID NO: 287), LASYLES (SEQ ID NO: 288), QHSRDLPLT (SEQ ID NO: 289), NYYMY (SEQ ID NO: 290), GINPSNGGTNFNEKFNK (SEQ ID NO: 291), and / or R. Antibodies comprising at least one of (SEQ ID NO: 292); MK3475; and / or blocking binders such as Keytruda may be included. In certain embodiments, the non-blocking binder, eg, antibody, is at least one of SEQ ID NOs: 17, 40, 63, 86, 109 and 132, or any one or more of SEQ ID NOs: 164-190. (Eg, SEQ ID NOs: 176 and 190) and / or derivatives thereof having at least one amino acid substitution may be included. In certain embodiments, the mammals are administered multiple times, which may be administered at intervals of about 1 week, about 2 weeks, about 3 weeks, or preferably about 1 month or more.

The present disclosure also provides a method for producing a binder described herein by expressing the binder in a cell and further isolating the binder from the cell or the culture supernatant of the cell. In some embodiments, the method may further comprise the step of expressing the nucleic acid encoding such a binder. In some embodiments, such methods may further comprise the step of combining the binder with one or more pharmaceutically acceptable excipients after isolation.

According to the present disclosure, a combination of binders such as a first binder that blocks the interaction between PD-1 and PD-L1 and a second binder that does not block the interaction between PD-1 and PD-L1. Also provided is a method of manufacturing. In some embodiments, the second binder binds PD-1. In some embodiments, the first and / or second binder is an antibody, such as, for example, a monoclonal antibody, or fragment or derivative thereof. In some embodiments, the second binder is SEQ ID NOs: 17, 40, 63, 86, 109, and 132 (eg 135C12); SEQ ID NOs: 2, 25, 48, 71, 94 and 117 (eg 139D6); It contains the amino acid sequences of SEQ ID NOs: 3, 26, 49, 72, 95, and 118 (eg 135D1); and / or SEQ ID NOs: 19, 42, 65, 88, 111, and 134 (eg 136B4). In some embodiments, these methods may further comprise the addition of pharmaceutically acceptable excipients.

The terms "about", "almost", etc., if preceded by a list of numbers or ranges, are within the list or range as if the term were placed immediately before each individual value in the list or range. Each individual value is referred to independently. These terms mean that the number it refers to is exactly the same as, close to, or similar to that number.

As used herein, "object" or "host" is meant to be an individual. Subjects include domesticated animals such as cats and dogs, livestock (eg cows, horses, pigs, sheep and goats), laboratory animals (eg mice, rabbits, rats, guinea pigs) and birds. You can stay. In one embodiment, the subject is a mammal, such as a primate or a human.

"Arbitrarily" or "arbitrarily" may or may not occur with or without the events or situations described subsequently, and the description indicates when the event or situation occurs and when it does not occur. Means to include. For example, the phrase "optionally a composition may contain a combination" is such that the composition is different such that the description includes both the combination and the non-existence of the combination (ie, the individual members of the combination). It means that it may or may not contain a combination of molecules.

In the present specification, the "range" can be expressed as from about one specific value and / or from about another specific value. When such a range is displayed, another aspect includes from one particular value to and / or the other specific value. Similarly, when a number is displayed as an approximation, it will be understood that the use of the antecedent "about" or "nearly" forms another aspect of that particular value. Furthermore, it will be appreciated that each endpoint of the range is significant both in relation to the other endpoints and independently of the other endpoints. The range (eg, 90-100%) is intended to include the range itself, as well as each independent value within that range, as if each number were listed individually.

The terms "combined," "in combination," or "in conjunction" are the physical combination of drugs administered together, or the treatment, prevention, and / or amelioration of a particular disease. Can mean the use of two or more agents in a treatment regimen for (eg, administered separately at physical and / or time).

When the terms "treat", "prevent" and / or "improve" are used in connection with a given treatment for a given condition (eg, to prevent infection by cancer or HIV). That the treated patient does not develop any clinically observable levels of the condition, or develops the condition more slowly and / or to a lesser extent than if he / she was not treated. Intended to convey. These terms are not limited to situations in which the patient does not experience any aspect of the condition. For example, "treatment" is given during a patient's exposure to a stimulant that is expected to give rise to a given manifestation of the condition, and less and / or milder than would be expected if it were not given. If the patient experiences a symptom, it will be said to have prevented the condition. For example, treatment can "prevent" the infection by causing the patient to show only mild and overt symptoms of the infection; this means that there should be no cell invasion by the infecting microorganism. It's not something to do.

Similarly, the "reduce," "reduce," and / or "reduce" used herein in connection with the prevention, treatment and / or amelioration of a given condition with a particular treatment is typically. Targets infections more slowly or to a lesser extent compared to control or basal levels that develop infections in the absence of treatment (eg, administration of one or more PD-1 binders). Means that occurs. Reducing the risk of infection can result in the patient exhibiting only mild overt symptoms of infection or delayed symptoms of infection; this means that there should be no invasion of cells by infecting microorganisms. It doesn't mean anything.

All references mentioned in this disclosure are incorporated herein by reference in their entirety. In the following examples, predetermined embodiments will be further described. These embodiments are provided by way of example only and are by no means intended to limit the scope of the claims.

Example 1
Preparation and characterization of PD-1 binding agents Four mouse strains (16 mice in total) were immunized with two PD-1 proteins, namely human PD-1 Fc fusion protein and human PD-1 monomer protein. Mice showing serum reactivity to PD-1 expressed on activated human T lymphocytes were selected to generate anti-PD-1 hybridoma cell lines. A total of 240 PD-1 hybridoma cell lines were selected to generate antibodies that bind to recombinant PD-1 protein. The primary criteria for first round antibody selection are: i) Staining PD-1 on activated human T lymphocytes by flow cytometry; ii) CDR VH and VL sequences compared to existing anti-PD-1 antibodies. And (iii) an epitope mapping performed by a competitive binding test using PD-1 conjugated Luminex beads with two commercially available anti-PD-1 antibodies that bind to different epitopes on PD-1. Second round selection is then: iv) affinity binding assay (not a primary criterion as it does not correlate with the stimulatory capacity of anti-PD-1 antibody); v) binds to PD-1 and Luminex biochemical. Evaluation of anti-PD-1 antibodies that are either blocking or non-blocking to PD-L1 binding in the assay; and vi) agonists (inability to recover T cells from functional exhaustion) or antagonists (T cells) Can be recovered from functional exhaustion) by functional property analysis of the antibody; In these tests, antibodies were tested and differentiated based on their ability to rescue proliferation in HIV-specific exhausted CD8 T cells.

An EFRA assay was performed to assess the functional effect of anti-PD-1 antibody on the proliferation of HIV-specific CD8 T cells in the primary screening of antibody supernatants from individual cell culture hybridoma cell clones. Antibody clones E8-3, C2-3, E1-3, F3-3, H8-3, C10-2, G2-1, G3-2, H2-1 and H4-2 act as PD-1 antagonists. Although found to stimulate proliferation, antibody clones C8-1 and G10-2 are agonistic (agonists) and promote the negative regulatory effects of PD-1. The level of growth induced by the peptide control (Pep 8) is slightly below 1% and the growth induced by the Merck MK-3475 anti-PD1 antibody is slightly above 2%. The antibodies of interest identified by these processes underwent a second round of subcloning and the resulting hybridoma clones were used for the production and purification of the antibodies shown in Table 3. A binding assay was performed with purified anti-PD-1 to confirm that the subclones retained their affinity for PD-1. Concentration-reactive binding of anti-PD-1 antibody to cell surface PD-1 was evaluated on activated CD4 T cells.

EFRA provides a selection of binders that restore T cells from functional exhaustion, but the selection interferes with the interaction of PD-1 with its biological ligand (eg, PD-L1 or PD-L2). Not necessarily limited to binders that do not. An embodiment of EFRA was to identify such a binder (antibody) that binds to PD-1. Epitope mapping of antibodies that bind to PD-1 was performed using two separate biochemical assays. In one assay, competitive binding to PD-1 Fc fusion protein-labeled beads was listed as one of two commercially available anti-PD-1 antibodies (clone EH12.2H7 and J116) (also also described in Table 3). Was evaluated with the anti-PD-1 antibody. Four classes of monoclonal antibodies that bind to distinct epitopes were identified based on this assay. They are: Class 1 (competitive with the commercially available monoclonal antibody clone EH12.2H7 that blocks the interaction of PD-1 and PD-L1), Class 2 (binding to PD-1 but with PD-1 and PD-L1). Competitive with the commercial monoclonal antibody clone J116, which does not effectively block the interaction of the Non-competitive). Antibodies were placed in one of four binding classes and the relative binding of these antibodies to cell surface PD-1 was expressed by mean fluorescence intensity (MFI) to control anti-PD-1 antibody. These results indicate that tightly bound antibodies were identified from all four binding classes. A second Luminex binding assay was used to directly assess whether the anti-PD-1 antibody blocked the interaction between PD-1 and PD-L1. This assay was performed with PD-1 Fc fusion protein coated beads, the PD-1 Fc fusion protein coated beads were incubated with the anti-PD-1 antibody from Table 3, followed by the PD-1 / antibody complex. Incubated with various concentrations of biotinylated PD-L1. The binding curve was compared to PD-L1 binding in the absence of competing anti-PD-1 antibody. It was found that one anti-PD-1 antibody from Table 3 (antibody 137F2 shown in FIG. 1A) can completely block the interaction between PD-1 and PD-L1. Antibodies listed in Table 3 as non-blocking antibodies for PD-1 / PD-L1 interactions result in a shift in PD-L1 binding affinity for PD-1, but mutual when PD-L1 concentrations increase. Does not block action (antibodies 135C12 and 136B4 shown in FIGS. 1B and C, respectively). In one interpretation, these antibodies are either non-competitive or partially competitive with the PD-1 / PD-L1 interaction. Some of these antibodies have also been shown to result in a statistically significant increase in proliferation compared to controls. Class 1 antibodies (competitive with the commercially available monoclonal antibody EH12.2H7, which blocks the interaction between PD-1 and PD-L1, or block in the PD-1 / PD-L1 interaction assay) are generally improved. It has been identified that it results in growth recovery. Combining data from multiple EFRA experiments and using MK-3475 as a common comparator, the selected antibodies listed in Table 2 are equivalent or statistically improved compared to the benchmark MK-3475 antibody. It showed the activity (p <0.007).

Example 2
Antibody Combinations It was found that combinations of antibodies that bind to different PD-1 epitopes enhance the recovery of HIV peptide-specific CD8 T cell proliferation in the functional exhaustion recovery assay. Synergies between antibody types were also observed. For example, it has been identified that class 1 (MK-3475) and class 2 (139D6) antibodies can simultaneously bind to PD-1. The highest irritation observed for MK-3475 is consistently about 200% for HIV peptide, while the combination of MK-3475 and 139D6 monoclonal antibodies at 5 μg / ml each is for HIV peptide control alone. HIV-specific CD8 T cell proliferation was increased to 288%, or proliferation was increased to 144% with respect to MK-3475 or 139D6 addition alone, demonstrating a synergistic effect. This synergistic increase in proliferation was observed with a statistically significant p-value of 0.007 in several experimental tests. By comparison, the addition of 10 μg / ml of either MK-3475 or 139D6 alone did not result in increased proliferation in EFRA. Therefore, a combination of binders, such as a first binder that blocks the interaction between PD-1 and PD-L1 and a second binder that does not block the interaction between PD-1 and PD-L1, is T. It has been identified as acting synergistically to rescue cells from exhaustion.

Example 3
Epitope Mapping of Anti-PD-1 Antibodies Preliminary epitope mapping evaluations are performed between commercially available anti-PD-1 antibodies (EH12.2H3 and J116) and the newly identified anti-PD-1 antibodies specified in this patent. It was based on a biochemical competitive binding assay test in. This procedure makes it possible to classify an antibody into one of four binding classes based on binding to PD-1 competitively or non-competitively with either of the above two commercially available antibodies. did. A more accurate method of identifying antibodies that bind to epitopes on PD-1 is to monitor antibody interactions with different PD-1 proteins that have careful amino acid substitutions at solvent-exposed residues. If these residues are important for tight binding of anti-PD-1 antibody, substitution reduces binding to PD-1 protein. These tests were performed by site-directed mutagenesis of the PD-1 gene encoded in the mammalian expression vector pReceiver-M67 under the control of the CMV promoter. Published structural data 3RQ PDBs for PD-1 proteins were used to design 31 different PD-1 clones with one, two or three amino acid substitutions at solvent-exposed residues. Residues involved in the interaction between PD-1 and PD-L1 / L2 were identified based on the open crystal structure of the complex of human PD-1 and PD-L2 (Lazar-Molnar, PNAS, 2008). , P10483-10488), and further mapped as a sphere numbered in the PD-1 structure shown in FIG. 2 (numbering corresponds to the amino acid residue of human PD-1 (FIG. 2H, SEQ ID NO: 275)). Substitution is at residues associated with PD-1 / PD-L1 interactions (represented by M10, M11, M12, M14, M23, M24, M25) or PDs not directly associated with PD-L1 interactions. -1 Residues on the opposite or adjacent surface of the protein (M1, M2, M3, M4, M5, M6, M7, M8, M9, M13, M15, M16, M17, M18, M19, M20, M21, (Represented by M22, M26, M27, M28, M29, M30, M31) and defined in FIG. HeLa cells were then transiently transfected with a Lipofectamine 2000 transfection reagent using a DNA vector encoding PD-1. Cells are incubated at 37 ° C. for 36-48 hours in a recruitment culture incubator to surface express PD-1 protein, then resuspend cells, from Table 3 between 0.3-2 μg / ml. Incubated for 30 minutes with a given anti-PD-1 antibody of choice. After the wash step, cells were stained with PE-labeled anti-mouse IgG secondary antibody and then analyzed by flow cytometry. In each experiment, the wild-type PD-1 protein was used as a positive control for antibody binding, and several antibodies that bind to different epitopes on PD-1 (is it blocking to PD-1 / PD-L1 interactions? Or by non-blocking (identified from competitive binding tests using commercially available PD-1 clones) in parallel to monitor relative expression levels of PD-1 proteins with different amino acid substitutions. did. All anti-PD-1 antibodies tested from Table 3 specifically bound to wild-type PD-1 transfected HeLa cells, but not to cells transfected with an empty vector control. All mutant PD-1 proteins are at near wild-type levels when stained with anti-PD-1 antibodies that are blocking or non-blocking to PD-1 / PD-L1 interactions. It resulted in a significant shift in mean fluorescence intensity compared to the expressed or untransfected controls. The selection set of antibodies from Table 3 was then systematically tested for their ability to bind to either wild-type or mutant PD-1 proteins expressed on the cell surface of transfected HeLa cells. These results are summarized in Table 11 showing the mutations that abolish or reduce binding for each of the various anti-PD-1 antibody clones. The flow cytometric histograms shown in FIGS. 4-8 provide examples of various antibodies that bind efficiently or with reduced affinity to HeLa cells transfected with the indicated mutant PD-1 construct. As expected, antibodies (MK-3475, 137F2, 140G5, and 139F11) that have been shown to be blocking PD-1 / PD-L1 interactions by biochemical assays are associated with that interaction site. It bound to the overlapping epitopes on PD-1. Amino acids (136B4, 135C12, 136E10) that are non-blocking to PD-1 / PD-L1 interactions, consistent with the biochemical PD-1 / PD-L1 interaction test, are PD-1 / PD-L1. It bound to a distinct epitope distal to the amino acid residue involved in the interaction. In FIG. 8, a significant shift in binding to the M17 PD-1 protein was observed for the 136B4 and 122F10 antibodies, whereas a slight shift in binding to the M26 PD-1 protein was observed for the 135C12 antibody. These differences clearly show that 136B4 and 135C12 have CDR loops that belong to separate families and both bind to different epitopes on PD-1 that overlap the P2 patch, as shown in FIG.

Example 4
Non-blocking antibody epitopes involved in the novel antagonist action of PD-1 Antibodies that bind to various PD-1 epitopes were evaluated for their antagonist activity in a functional exhaustion recovery assay. These tests have shown that antagonist antibodies can be either blocking or non-blocking to PD-1 / PD-L1 interactions. Considering that the mode of action due to the functional activity of the anti-PD-1 antibody is due to blocking of PD-1, these results show that PD-1 is inhibited by the non-blocking antagonist antibody. Imply new features. This claim is supported by the fact that the combination of blocking anti-PD-1 antibody and non-blocking anti-PD-1 antibody enhanced antagonist activity in the in vitro functional exhaustion recovery assay. This enhanced functional activity exceeds the induction levels that can be achieved with either antibody when administered alone and is a blocking (ie, competitive) anti-PD-1 antibody (MK3475 or 137F2). With non-blocking (ie, non-competitive or partially competitive in biochemical PD-1 / PD-L1 interaction assays) anti-PD-1 antibody 135C12 (FIG. 9A) or 139D6 (FIG. 9B). Demonstrated using various combinations. As further evidence of their functional activity, anti-PD-1 antibodies were tested alone or in combination with PD-1 / PD-L1 blocking and non-blocking antagonist antibodies in a mixed lymphocyte reaction assay (MLR). The MLR assay comprises mixing monocytes from one healthy donor with PD-1 ⁺ memory CD4 T cells from a second healthy donor. Monocytes were isolated from peripheral blood mononuclear cells (PBMC) by CD14 positive selection using magnetic beads. PBMCs from the second donor first depleted CD45RA-expressing cells with magnetic beads and then used a different set of magnetic beads for a positive selection of CD4-positive T cells. Next, these memory CD4 T cells (CD4 ^{+ CD45RA-} ) were subjected to AquaLive / Dead staining kit, as well as 127C2 anti-PD-1 antibody clones (with antagonist activity) for sorting based on viable PD-1 ⁺ cell population. It has not been shown to compete with MK3475, 137F2 or 135C12 for binding to PD-1) and was stained with a secondary anti-mouse PE antibody. After mixing monocytes from two different donors and CD4 ⁺ T cells in a ratio of 1: 2 or 1: 5, the cells are left untreated or shown in FIG. 10 at 1 μg / ml. Treated with anti-PD-1 antibody. After 5 days of incubation in the cell culture incubator, a portion of the cell culture supernatant was harvested and secreted IFNγ analyzed by ELISA. Tritium-labeled thymidine ( ³ H-TdR) was added to the cell culture medium and the cells were incubated for an additional 18 hours. ³ H-TdR uptake was used as an index of cell proliferation. This second independent in vitro assay confirmed the functional activity of our anti-PD-1 antibody. In FIG. 10a, up to 2-fold increased proliferation of CD4 ⁺ T cells was induced by individual or combination of anti-PD-1 antibodies. In FIG. 10b, a strong increase in IFNγ production was observed for all anti-PD-1 antibodies when tested individually, with 137F2 leading to the highest induction levels. Trends in increased proliferation of PD-1 ⁺ memory CD4 T cells and increased IFNγ production in combinations of blocking anti-PD-1 and non-blocking anti-PD-1 antibodies (either MK3475 and 135C12, or 137F2 and 135C12). Was observed. Increased IFNγ production from CD4 ⁺ T cells is associated with reduced pathology and improved memory in a mouse model of Alzheimer's disease when treated with PD-1 immune checkpoint blocking therapy (BaruchK, NatureMed, 2015). Given that, treatment with the anti-PD-1 antibody or anti-PD-1 antibody combination outlined therein is expected to have equivalent or improved therapeutic benefit. As described below, epitope mapping shows that all non-blocking antibodies with the highest potency bind to a similar patch of PD-1 (referred to herein as the "P2 patch"). The P2 patch contains regions of M4 amino acid substitutions (serine 38 to alanine, proline 39 to alanine, and leucine 41 to alanine) (FIG. 3; SEQ ID NO: 142)) and / or M17 amino acid substitutions (asparagine 102). Region of alanine and arginine 104 to leucine) (FIG. 3; SEQ ID NO: 155) and / or M18 amino acid substitution (asparaginate 105 to alanine) (FIG. 3; SEQ ID NO: 156) and / or M31. It overlaps the region of the amino acid substitution (leucine 41 to alanine and valine 43 to leucine) (FIG. 3; SEQ ID NO: 206).

To further investigate the region of PD-1 involved in the antagonist activity of anti-PD-1 antibody, solvent-exposed mutant amino acid residues from different species were mapped to human PD-1 structure. This structural representation based on the 3RQ PDB shows the plane of PD-1 interacting with PD-L1 / L2 in FIG. 11a and the opposite side of PD-1 in FIG. 11b. Different residues between monkey and human PD-1 are represented by red circles, and other mutant residues found in rodent (mouse and rat) PD-1 are represented by green circles, dog PD-. Additional mutant residues from 1 are shown in orange. Residues 46, 58, 74 and 116 are associated with the site of N-linked glycosylation and are marked with a turquoise circle. The alignment of the PD-1 ectodomain amino acid sequences of human, monkey, horse, dog, mouse and rat is shown in FIG. 11c, and the corresponding homology with human PD-1 is 96.6% and 79.9%, respectively. It is 73.2%, 62.4% and 66.4%. Structurally showing these interspecies mutant residues of PD-1 reveals that there are two conserved patches on PD-1 designated as P1 and P2 in FIGS. 11a and 11b. .. The P1 patch corresponds to the central region associated with the interaction between PD-1 and the PD-L1 / PD-L2 ligand (compare the residue marked with a sphere in FIG. 2 with that of FIG. 11a). .. Mutant residues that accumulate in PD-1 during evolution still need to maintain their interaction with PD-L1 / L2 ligands, so that this P1 patch is expected to be preserved between different species. Selective substitution of residues involved in PD-1 / PD-L1 interactions has been observed in mouse mutations (eg, residues 64 and 68), leading to co-evolution of PD-1 and its PD-L1 / L2 ligands. Although it can be caused; the interaction region of the core within P1 remains conserved. The evolutionarily conserved P2 patch occupies a surface region similar to the P1 patch, but this region does not have a role already identified for PD-1, both structurally and functionally. The evolutionary conservation of the P2 patch among the species examined indicates that PD-1 was under pressure to conserve the P2 site. This conservation is even more important given that only 62.4% to 79.9% are conserved in the four species examined by the PD-1 ectodomain. Considering all the mutant residues across these four species compared to human PD-1, there is an even lower 52.3% sequence conservation (FIG. 11c, blue arrows drawn in FIGS. 11a and 11b). Represents a structural area that exists). This functional pressure is due to the P2 patch acting as an interaction site for a ligand that has not yet been identified, or as a site for higher-order complex formation, or. It is still unclear if it is due to being associated with PD-1-related signaling events. However, the anti-PD-1 antibodies described therein, such as 135C12, 139D6, 135D1 and 136B4, which have binding epitopes that overlap the P2 patch, provide direct evidence of the functional importance of this region. ..

Given that this region has no pre-existing involvement in the functional activity of PD-1, the antibody represented by 135C12 having a binding that overlaps with the P2 patch on PD-1 shown in FIG. 11b is PD-. It is proposed here to explain the novel mechanism and site that exerts antagonistic activity on 1. Other antibodies, antibody fragments, or other protein binding agents capable of interacting with this P2 patch region of PD-1 are also PD-1 / PD-L1 as demonstrated in FIGS. 9, 10 and 17-20. It is further proposed to act as an antagonist of PD-1 in a manner distinct from and complementary to anti-PD-1 antibodies that act through blockade of interactions. As a further example of the importance of the P2 patch region in PD-1, the 136B4 antibody binds to a separate epitope on PD-1 as compared to the antibodies 135C12, 139D6 and 135D1 which also overlap with the P2 patch. 136B4 is associated with M17 and M18 PD-1 mutations (M17: asparagine 102 to alanine and arginine 104 to alanine, M18: aspartic acid 105 to alanine) located at the edge of the P2 patch. Shows strongly reduced affinity. Along with the 135C12, 139D6 and 135D1 antibodies, 136B4 showed the highest functional activity of all tested non-blocking antibodies.

Example 5
Antibody Competitive Binding Tests To further characterize the binding epitopes of the selected antibodies listed in Table 3, a series of antibody competitive binding tests were performed for binding to cell surface PD-1. A Maxpar® antibody labeling kit (Fluidigm) was used to chemically conjugate to antibodies (MK3475, 137F2, 135C12 and 134D2) that bind to distinct epitopes on PD-1. In this procedure, the disulfide bond of the antibody is partially reduced in order to bind the cysteine residue to the metal chelate polymer. Next, using these metal isotope-modified antibodies, cells were labeled to see the expression level of surface PD-1, and analysis was performed by mass cytometry. Peripheral blood mononuclear cells from healthy donors were stimulated with PHA and IL-2 and incubated for an additional 5 days to generate CD4 T cells with high levels of PD-1 expression. After cell staining, gating was performed to select surviving CD3 ⁺ / CD4 ⁺ cells, and the cell surface level of PD-1 was evaluated for each sample by mass cytometry. In an antibody competition assay, a sample of ^2x106 cells was incubated with one of the indicated competing antibodies shown in FIG. 12 at 15 μg / ml at 4 ° C. for 40 minutes. The metal isotope-labeled stained anti-PD-1 antibody (MK3475, 137F2, 135C12, or 134D2) was then added to the cells at <1 μg / ml, incubated for an additional 20 minutes, then washed to give the unbound antibody. It was removed and further analyzed for cell surface PD-1 levels. Using mouse IgG1 isotype control antibodies as negative controls and as a reference (reference), the percentage of PD-1 positive cells for the sample incubated with anti-PD-1 competing antibody clones was calculated.

All Class 1 anti-PD-1 antibodies that are blocking PD-1 / PD-L1 interactions were competitive in experiments in which PD-1 hypercells were stained with either MK3475 or 137F2 (to IgG1 controls). On the other hand, with ≦ 29% staining of PD-1 positive cells (as shown in FIG. 12). Given that the epitope mappings of some of these antibodies have binding sites that are identical or overlap, these results are predictable. Class 1 antibodies were also competitive or partially competitive with 134D2 antibodies (as shown in FIG. 12 with 30-59% staining relative to the IgG1 control). 134D2 is blocking the PD-1 / PD-L1 interaction, but binds primarily to epitopes that overlap with the M15 mutation but to a lesser extent with the M17 mutation. Class 1 antibodies were found to be non-competitive with 135C12 antibodies (as shown in FIG. 12 with ≧ 60% PD-1-positive cell staining (binding of labeled antibody) to IgG1 controls). , Supporting an epitope binding test showing that these antibodies bind to distinct sites on PD-1.

Using class 2 antibodies as competitors, both MK3475 and 137F2 labeled antibodies bind with little or no reduction in binding compared to IgG1 controls. One exception is the 135D1 antibody, which blocked PD-1 binding of labeled 137F2. Given that the epitope of 135D1 is close to the M4 mutation, there appears to be some steric hindrance to 137F2 antibody binding in the M23 mutation region. Given that 135C12 and 139D6 clones have a shift in binding to PD-1 encoding the M4 mutation, but they do not compete with binding to 137F2, binding of these class 2 antibodies is more than in the P2 patch region. In the center. The 135C12 antibody also had a small shift in the M17, M18, M26, M28, and M31 mutations, all found at the edges of the P2 patch. All Class 2 antibodies used as competitors blocked binding of label 135C12 and confirmed that they bound to overlapping epitopes on PD-1. Most Class 2 competing antibodies do not compete with label 134D2 for binding to PD-1. The 136B4 antibody is an exception to class 2 antibodies, which block the binding of 134D2. These results are consistent with the epitope binding test in Example 3, where binding of 134D2 to PD-1 is partially disrupted by the M17 mutation, but predominantly by the M13 and M15 mutations. Binding of 136B4 antibody is blocked only by M17 and M18 mutations within the edge of the P2 patch or within the P2 patch.

Class 3 antibodies are competitive or partially competitive with MK3475 and 137F2 (defined as 30-59% staining relative to IgG1 control, as shown in FIG. 12) and non-135C12. Competitively binds to PD-1. The 135E10 class 3 antibody is competitive with the labeled 134D2, whereas the 140A1 class 3 antibody is non-competitive with the labeled 134D2. Epitope mapping shows that 140A1 binds at a region of M13 mutation that is different from the binding epitope of 134D2 (mainly M15 and mildly M17).

In the evaluation of functional data for Class 4 antibodies, only 134D2 and 136B5 have significant antagonist activity leading to increased proliferation of HIV-specific CD8 T cells in the functional exhaustion recovery assay (as shown in FIG. 12). Increased proliferation of 150%). These antibodies also block the interaction of PD-1 with PD-L1 in biochemical assays and therefore function as antagonists, presumably via PD-1 / PD-L1 blockade. Excess 136B5 blocks PD-1 ⁺ cell staining with label 134D2, so these antibodies bind to PD-1 with similar or overlapping epitopes (FIG. 12). Competitive binding data with the remaining Class 4 antibodies shows that they bind to various sites on PD-1 and that this binding generally does not overlap with the 135C12 antibody. The 122F10 antibody is an exception that binds to an epitope similar to or overlaps with 136B4. Because these two antibodies have similar binding profiles in antibody competition tests (FIG. 12), both have reduced binding to the M17 mutant PD-1 construct (Table 11).

PD to which various anti-PD-1 antibody clones bind by combining information from the epitope mapping test (Example 3) and antibody competition test (FIG. 12) performed by amino acid substitution at the solvent-exposed residue of PD-1. A consistent structural map of the region on -1 is shown in FIGS. 13a and 13b. These binding epitopes are shown in Table 11 as circular patches of different colors for most clones. FIG. 13a shows the plane of PD-1 with the majority of residues associated with PD-1 / PD-L1 or PD-1 / PD-L2 interactions. The exceptions are residues 89 and 90 found on the left side of FIG. 13a in the flexible loop region. The first set of antibodies that compete with the PD-1 / PD-L1 interaction, such as clones 137F2, 139F11, 140A1, 140G5 and MK3475, all bind to this aspect of PD-1. The two antibodies identified as having the highest antagonist activity in the functional assay (Table 11) are both shown in FIGS. 11a and 13a and are the center of the P1 patch, concentrating on residues 124-126 of PD-1. Note that it binds to. This finding is consolidated by the fact that 137F2 and 139F11 have significantly different heavy and light chain CDR sequences and belong to distinct families based on the sequence alignment of all antibody clones studied herein. Be done. Antibody clones 134D2 and 136B5 have been shown to compete with PD-1 / PD-L1 interactions in biochemical assays, but they overlap with the M13, M15 and M17 mutations, FIG. 13b. It binds to the region of PD-1 shown in. Based on this mapped epitope, these antibodies bind near residues 85 and 86 of PD-1 that are part of the loop region on PD-1 containing residues 89 and 90. Since residues 89 and 90 are associated with interaction with PD-L1 (FIG. 2a), binding to this loop region induces structural changes in PD-1 that inhibit binding to PD-L1. sell. Consistent with these results, antibody competition tests show that the addition of competing class 1 antibody significantly reduces binding of the labeled 134D2 antibody (FIG. 12).

All antibodies that are non-blocking to the D-1 / PD-L1 interaction listed in Table 3 bind to epitopes away from the residues associated with the PD-1 / PD-L1 interaction. All antibodies with strong functional activity (CD8 T cell proliferation enhanced to> 150% in the functional recovery assay, FIG. 12) bind in epitopes that overlap the P2 patch shown in FIG. 13b. The 135C12 (all test mutants), 139D6 (selected test mutants), and 135D1 (selected test mutants) antibody clones have associated heavy and light chain CDR sequences and It binds to sites that overlap with the M4, M18 and M31 mutations. The 136B4 antibody clone has a distinct CDR sequence and shows a large shift in binding affinity when using the M17 and M18 mutant PD-1, which binds to the distinct epitope at the edge of the P2 patch. Show that. The 135C12 antibody competitively binds to 136B4 claws but does not compete with either the 134D2 or 136E10 clones. These antibody competitive binding tests further support binding of 135C12, 139D6135C1, and 136B4 to epitopes that overlap the P2 patch. The binding epitope of the 136E10 antibody is limited to the M1 mutation that completely abolishes the binding of this antibody to PD-1.

Example 6
Targeted killing by antibody-drug conjugates As discussed herein, binders such as antibodies (eg, Table 3) are conjugated with toxins to perform targeted killing of PD-1-expressing cells. You may gate it. Examples of therapeutic benefits of this strategy include targeted exclusion of HIV-infected CD4 ⁺ T cells, which are predominantly PD-1 positive. To carry out these tests, two different anti-PD-1 antibodies (140G5 and 136B5) were PEG4-vc-PAB-PNU- via the available cysteine group after partial reduction of the antibody's disulfide bond. It was chemically conjugated with 159682 (hereinafter referred to as PNU). The antibody drug conjugate (ADC) was profiled by hydrophobic interaction chromatography and size exclusion chromatography and both samples were found to contain> 95% PNU conjugated antibody. In the ADC killing assay, CD4 ⁺ T cells were isolated from PBMCs of patients chronically infected with the HIV-1 virus. The CD4 ⁺ T cells were incubated with an isotype control antibody, 140G5 mAb, 140G5-PNU mAb conjugate, 136B5 mAb, or 136B5-PNU mAb conjugate. All antibody concentrations were 10 μg / ml and cells were incubated at 37 ° C. for 5 days in a 5% CO ₂ cell culture incubator. On day 5, in addition to Aqua staining for cell viability and Annexin V staining to identify cells undergoing apoptosis, antibodies were used to monitor cell surface levels of CD4 and PD-1. The cells were stained for flow cytometric analysis. As shown in FIG. 14, all samples treated with various antibodies or ADCs are equivalent Aqua positive (FIG. 14a) or Annexin V positive in CD4 ⁺ T cells with low to moderate levels of expression of PD-1. (Fig. 14b) has a level. However, in cell populations with high levels of PD-1, the amount of Annexin V-positive and Aqua-positive cells in samples treated with anti-PD-1 ADC (140G5-PNU and 136B5-PNU in FIGS. 14a and 14b). There is a significant increase.

To provide further evidence of the potential therapeutic benefit of anti-PD-1 ADCs, various antibodies or ADCs (isotype control antibodies, 140G5 mAbs, 140G5-PNU mAb conjugates, 136B5 mAbs, or 136B5-PNU mAb conjugates). CD4 ⁺ T cells treated at the gate) for 5 days were evaluated for the frequency of HIV-infected cells using a quantitative virus outgrowth assay. In this assay, cells from HIV-infected patients treated with antibody or ADC are left unstimulated or stimulated with anti-CD3 / anti-CD28 antibody and then in the presence of allogeneic CD8-depleted PBMC. Cultured at various dilutions. By testing with CD4 ⁺ T cells at various dilutions from each sample, the frequency of cells containing replicable virus can be assessed after 5 days of treatment with antibody or ADC. After 14 days of incubation of CD4 ⁺ T cells and allogeneic CD8-depleted PBMCs, they were tested in p24 ELISA and for the presence of HIV RNA to determine the frequency of cells containing replicable virus. According to the specific depletion of PD-1 hypercells shown in FIG. 14, an increase in the proportion (%) of Annexin V-positive or Aqua-positive PD-1 hypercells was observed, and the cells undergoing apoptosis and cell death, respectively. Shows an increase (FIGS. 15a and 15b). CD4 ⁺ T from HIV-infected donors treated with or untreated with IgG1 isotype control antibody, anti-PD-1 antibody (136B5 or 140G5) for 5 days in the virus outgrowth assay shown in FIG. All cell samples have a frequency of cells containing a similar replicable virus (16-28 HIV-1 RNA-positive cells / ml (RUPM)). However, in samples treated with anti-PD-1 ADC (140G5-PNU mAb conjugate, 136B5-PNU mAb conjugate, or 136B5-PNU mAb conjugate), infections consistent with a 4-5-fold reduction in cells capable of producing infectious virus. There is a marked decrease in cell frequency. These results have been reproduced in several other experiments with a 10-fold reduction in RUPM associated with anti-PD-1 ADC treatment for 5 days. Therefore, these studies provide in vitro proof of the idea that anti-PD-1 ADCs can be an effective treatment for depleting infected cells from HIV-1 positive patients.

Example 7
Humanization of Mouse Anti-PD-1 Antibodies To find heavy and light chain frameworks that are most similar to those of mouse antibodies, select mouse IgG1 antibody sequences from anti-PD-1 clones with the human immunoglobulin germline V gene. Compared to the database. Using these framework sequences, a combinatorial library was designed and used to construct a phage display library incorporating the heavy and light chain CDR loops of the antibody to be humanized. The phage library was then used in panning experiments against recombinant human PD-1 in the _Fc fusion construct. Phage ELISA was used to evaluate humanized anti-PD-1 output phage that bind to recombinant PD-1. _Fab fragment DNA from positive clones was introduced into the FASEBA (fast screening for expression, biophysical-properties and affinity) library and used to generate _Fab protein fragments. These _Fab fragments were evaluated for expression levels, protein stability / biophysical properties, and affinity for recombinant human _Fc -PD-1 proteins as identified by the Biacore test. The VH and VL sequences obtained for humanized 137F2 and 135C12 mouse antibody clones of Table 3 in this manner and with the desired expression, stability and affinity properties are shown below. The results of Biacore affinity measurement of 137F2 and 135C12 humanized _Fab clones for human and monkey F _c -PD-1 proteins are shown in Tables 12 and 13, respectively. The variable region amino acid sequences of these exemplary humanized antibodies (heavy and light chains as indicated) are shown below.

A. Humanized 137F double chain sequence 1. Mouse VH reference sequence QVQLQPGAELVRPGTSVKMSCKAAGYTFTNYWIGWIKQRPGHGLEWIGDIYPGGGYTNYNEYNEKFKGKATTLTADTSSSTAYMQVSSLTSEDTGIYYCARGYDFVLDRWGQGTS

2. 2. A35790-VH
QMQLVQSGPEVKKPGTSVKVSCKASGFFTNYWIGWVRQAPGQUAREWMGDIYPGGGYTNYNEKFKGRVTITADKSTSTAYMELSSLDTRVYYCARGYDFVLDRWGQGTTVSS (SEQ ID NO: 139)

3. 3. A35796-VH
QMQLVQSGPEVKKPGTSVKVSCKASGFFTNYWIGWVRQAPGQUAREWMGDIYPGGGYTNYNEKFKGRVTITADKSTSTAYMELSSLDTRVYYCARGYDFVLDRWGQGTTVSS (SEQ ID NO: 140)

4. A35793-VH
QMQLVQSGPEVKKPGTSVKVSCKASGFFTNYWIGWVRQAPGQUAREWMGDIYPGGGYTNYNEKFKGRVTITADKSTSTAYMELSSLDTRVYYCARGYDFVLDRWGQGTTVSS (SEQ ID NO: 14)

5. A35518-VH
QMQLVQSGPEVKKPGTSVKVSCKASGFFTNYWIGWVRQAPGQUAREWMGDIYPGGGYTNYNEKFKGRVTITADKSTSTAYMELSSLDTRVYYCARGYDFVLDRWGQGTTVSS (SEQ ID NO: 142)

6. A35795-VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWIGWVRQUAPGQGLEWMGDIYPGGGYTNYNEKFKGRVTITADKSTSTAYMELLSSLDTAVYYCARGYDFVLDRWGQGTTVSS

7. A35797-VH
EVQLVQSGAEVKKHGESLKISCKGSYSFTNYWIGWVRQATGQGLEWMGDIYPGGGYTNYNEKFKGRVTITADKSTSTAYMELLSSLDTAVYYCARGYDFVLDRWGQGTTVSS4

8. A35799-VH
QMQLVQSGAEVKKTGSSVKVSCKASGYTFTNYWIGWVRQMPGKGLEWMGDIYPGGGYTNYNEKFKGRVTITADKSTSTAYMELLSSLDTAVYYCARGYDFVLDRWGQGTTVSS

9. A35805-VH
QVQLVQSGSELKKPGASVKVSCKASGYTFTNYWIGWVRQAPGKGLEWMGDIYPGGGYTNYNEKFKGRVTITADKSTSTAYMELLSSLDTAVYYCARGYDFVLDRWGQGTTVSS

10.137F VH1
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWIGWVRQUAPGQGLEWMGDIYPGGGYTNYNEKFKGRGTMTRDTSTSTVYMELLSSLDTAVYYCARGYDFVLDRWGQGTRLVTVSS

11.137F VH2
QVQLVQSGAEVKKPGASVKVSCKAAGYTFTNYWIGWVRQAPGQGLEWMGDIYPGGGYTNYNEYNEKFKGRVTMTADTSTSTVYMELSSLSREDTAVYYCARGYDFVLDRWGQGTLVTVSS

12.137F VH1b
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWIGWVRQUAPGQGLEWIGDIYPGGGYTNYNEYNEKFKGRVTMTADTSTSTVYMELSSLSREDTAVYYCARGYDFVLDRWGQGTLVTVSS

13.137F VH1c
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWIGWIRQAPGQGLEWIGDIYPGGGYTNYNEKFKGRATTLTADTSTSTVYMEVSSLRSDATEVYYCARGYDFVLDRWGQGTLVTVSS

B. Humanized 137F2 light chain sequence 1. Mouse VL reference sequence DIVMSQSSLAVSTGEKVTMTCKSQSLFNSETQKNYLAWYQQKPGQPKLLIFWASTRESGVPDRFLLGSGSGTDFTLTISSVQAEDLAVYYCKQSYTLRTFGGTKLEIK (sequence number 2)

2. 2. A35790-VL
DVMTQSPAFLSVTPGEKVTITCKSSQSLFNSETQKNYLAWYQQKPGQPPPKLLIYWASTRESGVPSRFSGSGSGTDFTLTSSLQPDFATTYCKQSYTLRTFGGTKLEIK (SEQ ID NO: 151)

3. 3. A35796-VL
DVMTQSPAFLSVTPGEKVTITCKSSQSLFNSETQKNYLAWYQQKPGQPPPKLLIYWASTRESGVPSRFSGSGSGTDFTLTSSLQPDFATTYCKQSYTLRTFGGTKLEIK (SEQ ID NO: 152)

4. A35793-VL
DVMTQSPAFLSVTPGEKVTITCKSSQSLFNSETQKNYLAWYQQKPGQPPPKLLIYWASTRESGVPSRFSGSGSGTDTFFTISSLEAEDAATYCKQSYTLRTFGGTKLEIK (SEQ ID NO: 153)

5. A35518-VL
DVMTQSPAFLSSVTPGEKVTITCKSSQSLFNSETQKNYLAWYQQKPGQAPRLLLYWASTRESGVPSRFSGSGSGTDFTFTISLEAEDAAATTYCKQSYTLRTFGGTKLEIK (SEQ ID NO: 154)

6. A35795-VL
DVMTQSPAFLSVTPGEKVTITCKSSQSLFNSETQKNYLAWYQQKPGQAPRLLLYWASTRESGVPSRFSGSGSGTDFTLTISSSLQPDFATTYCKQSYTLRTFGGTKLEIK (SEQ ID NO: 15)

7. A35797-VL
DIQMTQSLSLSVSVGDRVTITCKSSQSLFNSETQKNYLAWYQQKPGQPQPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSSLQPDFATTYCKQSYTLRTFGGTKLEIK (SEQ ID NO: 156)

8. A35799-VL
DVMTQSPAFLSVTPGEKVTITCKSSQSLFNSETQKNYLAWYQQKPGQPPPKLLIYWASTRESGVPSRFSGSGSGTDFTLTSSLQPDFATTYCKQSYTLRTFGGTKLEIK (SEQ ID NO: 157)

9. A35805-VL
DVMTQSPAFLSVTPGEKVTITCKSSQSLFNSETQKNYLAWYQQKPGQPPPKLLIYWASTRESGVPSRFSGSGSGTDFTFSSLEAEDAAATTYCKQSYTLRTFGGTKLEIK (SEQ ID NO: 158)

10.137F VL1
DIVMTQSPDSLAVSLGERATINCKSSQSLFNSETQKNYLAWYQQKPGQPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSSLQAEDVAVYYCKQSYTLRTFGQGTKLEIK (SEQ ID NO: 159)

11.137F VL2
DIVMTQSPDSLAVSLGERATTITCKSSQSLFNSETQKNYLAWYQQKPGQPKLLIFWASTRESGVPDRFLGSGSGSGTDFTLSSLQAEDVAVYYCKQSYTLRTFGQGTKLEIK (SEQ ID NO: 160)

12.137F VL1b
DIVMTQSPDSLAVSLGERATINCKSSQSLFNSETQKNYLAWYQQKPGQPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSSLQAEDVAVYYCKQSYTLRTFGQGTKLEIK (SEQ ID NO: 161)

13.137F VL1c
DIVMTQSPDSLAVSLGERATTITCKSSQSLFNSETQKNYLAWYQQKPGQPKLLIFWASTRESGVPDRFSGSGSGTDFTLTISSSLQAEDVAVYYCKQSYTLRTFGQGTKLEIK (SEQ ID NO: 162)

14.137F VL1d
DIVMTQSPDSLAVSLGERATMTCKSSQSLFNSETQKNYLAWYQQKPGQPKLLIFWASTRESSGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCKQSYTLRTFGQGTKLEIK (SEQ ID NO: 163)

C. Humanized 135C12 heavy chain sequence 1. Mouse VH reference sequence EVQLHQSGPELLKPGASVRMSCKASGYTFTNFYIHWVKQSHGKSIEWIGSYPNYGDTAYNQKFKDKATLTVDKSSSTAYMALRSLTSEDSAVYYCARGYSYAMDYWG

2. 2. A35775-VH
EVQLVQSGAEVKKPGESSLKISCKGSGYSFTNFYIHWVRQAPGQRLEWMGSIYPNYGDTAYNQKFKDRFVFSLDTVSTAYLQISSLKAEDTAVYYCARGYSYAMDYWGQGTTVSS

3. 3. A35783-VH
EVQLVQSGAEVKKPGESSLKISCKGSGYSFTNFYIHWVRQARGQRLEWIGSIYPNYGDTAYNQKFKDRFVFSLDTVSTAYLQISSSLKAEDTAVYYCARGYSYAMDYWGQGTTVSS

4. A35774-VH
EVQLVQSGAEVKKPGESSLKISCKGSGYSFTNFYIHWVRQAPGQRLEWMGSIYPNYGDTAYNQKFKDRFVFSLDTVSTAYLQISSLKAEDTAVYYCARGYSYAMDYWGQGTTVSS

5. A36443-VH
EVQLVQSGAEVKKPGBATVKISCCKVSGYTFTNFYIHWVRQARGQRLEWIGSIYPNYGDTAYNQKFKDRVTITADKSTSTAYMERSSLDTAVYYCARGYSYAMDYWGQGTTVSS (SEQ ID NO: 16)

6. A35777-VH
EVQLVQSGAEVKKPGBATVKISCCKVSGYTFTNFYIHWVRQAPGKGLEWMGSIYPNYGDTAYNQKFKDRVTITADKSTSTAYMELSSLDTAVYYCARGYSYAMDYWGQGTTVSS (SEQ ID NO: 16)

7. A35789-VH
EVQLVQSGAEVKKPGESSLKISCKGSGYSFTNFYIHWVRQMPGKGLEWMGSIYPNYGDTAYNQKFKDRVTITADKSTSTAYMELSSLDTAVYYCARGYSYAMDYWGQGTTVSS

8. A36448-VH
EVQLVQSGAEVKKPGESSLKISCKGSGYSFTNFYIHWVRQMPGKGLEWMGSIYPNYGDTAYNQKFKDRFVFSLDTVSTAYLQISSLKAEDTAVYYCARGYSYAMDYWGQGTTVSS

9. A36437-VH
EVQLVQSGAEVKKPGESSLKISCKGSGYSFTNFYIHWVRQMPGKGLEWMGSIYPNYGDTAYNQKFKDRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARGYSYAMDYWGQGTTVSS

10.135C VH1
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNFYIHWVRQAPGQGLEWMGSIYPNYGDTAYNQKFKDRVTMTRDTSTSTVYMELLSSLDTAVYYCARGYSYAMDYWGQGT

11.135C VH2
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNFYIHWVRQUAPGQGLEWMGSIYPNYGDTAYNQKFKDRVTMTVDKSTSTVYMELLRSLRSEDTAVYYCARGYSYAMDYWGQGT

12.135C VH3
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNFYIHWVKQAHGQGLEWMGSIYPNYGDTAYNQKFKDRVTMTVDKSTSTVYMELLRSLRSEDTAVYYCARGYSYAMDYWGQGT

13.135C VH1b
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNFYIHWVRQAPGQGLEWIGSIYPNYGDTAYNQKFKDRVTMTVDTSTSTVYMELSSLDTAVYYCARGYSYAMDYWGQGTLVTVSS

14.135C VH1c
QVQLVQSGAEVKKPGASVKMSCKASGYTFTNFYIHWVRQUAPGQGLEWIGSIYPNYGDTAYNQKFKDRATLTVDTSTSTAYMELSSLDTAVYYCARGYSYAMDYWGQGTLVTVSS (SEQ ID NO: 17)

15.135C VH1d
QVQLVQSGAEVKKPGASVKMSCKASGYTFTNFYIHWVRQUAPGQGLEWIGSIYPNYGDTAYNQKFKDRATLTVDKSTSTAYMELSSLDTAVYYCARGYSYAMDYWGQGTLVTVSS

D. Humanized 135C12 light chain sequence 1. Mouse VL reference sequence NIVMTQSDKSMSVGERVTLTCKASSENVVTYVSWYQQKPEQSPKLLIYGASNRYTGVPDRFTGSGSATDFTLTISSVQAEDLADYHCGQGYSYPYTFGGGTKLEIK (SEQ ID NO: 280)

2. 2. A35775-VL
DIQMTQSPSVSSASVGDRVTITCSASSQGISGDLNWYQQKPGQAPRLLLIYHTSSSLHSGVPSRFSGSGSGSTDFTLTISSSLQPEDFATYYCQYYSKDLLTFGGTKLEIK (SEQ ID NO: 178)

3. 3. A35783-VL
DIQMTQSLSLSVSVGDRVTITCSASSQGISGDLNWYQQKPGKTPKLLIYHTSSSLHSGVPSRFSGSGSGSTDFTSSLQPEDFATYYCQYYSKDLLTFGGTKYYCQYYSKDLLTFGGTKLEIK (SEQ ID NO: 179)

4.35774-VL
DIQMTQSPSVSSASVGDRVTITCSASSQGISGDLNWYQQKPGKAPKLLIYHTSSSLHSGVPSRFSGSGSGTDFTLTISSSLQPEDFATYYCQYYSKDLLTFGGTKLEIK (SEQ ID NO: 180)

5. A36443-VL
DIQMTQSPSVSSASVGDRVTITCSASSQGISGDLNWYQQKPGQAPRLLLIYHTSSSLHSGVPSRFSGSGSGTEFTLTISRLEPEDFAVYYCQYYSKDLLTFGGTKLEIK (SEQ ID NO: 181)

6. A35777-VL
DIQMTQSPSTLSASVGDRVTITCSASSQGISGDLNWYQQKPGQAPRLLLIYHTSSSLHSGVPSRFSGSGSGSTDFTLTISSSLQPEDFATYYCQYYSKDLLTFGGTKLEIK (SEQ ID NO: 182)

7. A35789-VL
DIQMTQSPSVSSASVGDRVTITCSASSQGISGDLNWYQQKPGQAPRLLLIYHTSSSLHSGIPARFSGSGSGTDFTLTISRLEPEDFAVYYCQYYSKDLLTFGGTKLEIK (SEQ ID NO: 183)

8. A36448-VL
DIQMTQSPSTLSASVGDRVTITCSASSQGISGDLNWYQQKPGKTPKLLIYHTSSSLHSGVPSRFSGSGSGSTDFTLTISSSLQPEDFATYYCQYYSKDLLTFGGTKLEIK (SEQ ID NO: 184)

9. A36437-VL
DIQMTQSPSTLSASVGDRVTITCSASSQGISGDLNWYQQKPGQAPRLLLYHTSSLHSGIPARFSGSGSGTDFTLTISSSLQPEDVABYYCQYYSKDLLTFGGTKLEIK (SEQ ID NO: 185)

10.135C VL1
DIQMTQSLSLSVSVGDRVTITCSASSQGISGDLNWYQQKPGKAPKLLIYHTSSSLHSGVPSRFSGSGSGSTDFTSSLQPEDFATYYCQYYSKDLLTFGGTKYYCQYYSKDLLTFGGTKLEIK (SEQ ID NO: 186)

11.135C VL2
DIQMTQSLSLSVSVGDRVTITCSASSQGISGDLNWYQQKPGKAVKLLIYHTSSSLHSGVPLRFSGSGSGTDYTLTISSSLQPEDFATYYCQYYSKDLLTFGGTKLEIK (SEQ ID NO: 187)

12.135C VL3
DIQMTQSLSLSVSVGDRVTITCSASSQGISGDLNWYQQKSDGAVKLLIYHTSSSLHSGVPLRFSGSGSGTDYTLTISSSLQPEDFATYYCQYYSKDLLTFGGTKLEIK (SEQ ID NO: 188)

13.135C VL1b
DIQMTQSLSLSVSVGDRVTITCSASSQGISGDLNWYQQKPGKAPKLLIYHTSSSLHSGVPSRFSGSGSGSTDYTLTISSSLQPEDFATYYCQYYSKDLLTFGGTKLEIK (SEQ ID NO: 189)

14.135C VL1c
DIQMTQSLSLSVSVGDRVTITCSASSQGISGDLNWYQQKPGKAVKLLIYHTSSSLHSGVPSRFSGSGSGTDYTLTISSSLQPEDFATYYCQYYSKDLLTFGGTKLLK (SEQ ID NO: 190).

The results of the Biacore binding affinity measurement of the indicated humanized antibody are shown in Tables 12 and 13 below.

Example 8
Stimulation of T cells with Anti-PD1 Antibodies and Combinations The results of antigen-specific stimulation of PBMCs from chronically infected HIV donors that result in HIV-specific CD8 ⁺ T cell proliferation are shown in FIGS. 17-19. Due to the exhaustion of CD8 ⁺ T cells in viremia donors, the addition of anti-PD-1 antibody enhances antigen-specific proliferation by almost 200% (p-value <0.0001).

Humanized anti-PD-1 antibodies encoding CDRs derived from mouse blocking anti-PD-1 antibody 137F2 and mouse non-blocking anti-PD-1 antibody 135C12 were subjected to functional exhaustion in antigen-specific CD8 T cells in a CFSE functional exhaustion assay. Evaluated for their ability to rescue. Several humanized antibodies of 137F2 and 135C12 clones with variants in the human antibody framework region were tested in multiple assays, all with MK3475, and 137F2 and 135C12 mouse / human chimeric antibodies (V from mice). It showed similar activity in promoting CD8 ⁺ T cell proliferation as compared to the _L and _VH moieties, and the human constant region of IgG4). Due to the exhaustion of CD8 T cells in viremia donors, the addition of anti-PD-1 antibody enhances antigen-specific proliferation by almost 200% (p-value <0.0001) (FIG. 17). Mouth blocking anti-PD-1 antibody (ie, blocking PD-1 / PD-L1 interaction) 137F2-derived CDR-encoding humanized anti-PD-1 antibody (ie, humanized antibody A35795 (ie, SEQ ID NOs: 143 and 155). Includes), A35796 (including SEQ ID NOs: 140 and 152) and A35797 (including SEQ ID NOs: 144 and 156), and Mouse-Human Chimera 137F2 (including SEQ ID NOs: 8, 31, 54, 77, 100, and 123). , And humanized anti-PD-1 antibodies encoding CDRs derived from the mouse non-blocking anti-PD-1 antibody 135C12 (ie, including humanized antibodies A35774 (including SEQ ID NOs: 166 and 180), A35783 (including SEQ ID NOs: 165 and 179). ), A35789 (including SEQ ID NOs: 169 and 183), A36443 (including SEQ ID NOs: 167 and 181), 135C H2L2 (including SEQ ID NOs: 173 and 187), 135C H1cL1c (including SEQ ID NOs: 176 and 190), 135C H1cL1b. Functions in antigen-specific CD8 ⁺ T cells (including SEQ ID NOs: 176 and 189), and mouse-human chimera 135C12 (including SEQ ID NOs: 17, 40, 63, 86, 109 and 132)) in a CFSE functional exhaustion assay. We evaluated their ability to relieve fatigue. Several humanized antibodies of 137F2 (blocking) and 135C12 (non-blocking) clones with variants in the human antibody framework region were tested in multiple assays. All showed similar activity in promoting CD8 ⁺ T cell proliferation compared to MK3475, as well as mouse / human chimeric antibodies of 137F2 and 135C12.

FIG. 18 shows the results of a combination of an antibody that blocks PD-1 / PD-L1 interactions (MK3475 or A35795) and a non-blocking anti-PD-1 antibody (A35774) compared to either antibody alone. It is shown to have resulted in a statistically significant (p-value <0.0001) increase in proliferation. The data shown in FIG. 18 is the average of 3 experiments for the combination of MK3475 and A35774 and the average of 4 experiments for the combination of A35795 and A35774.

FIG. 19 shows an antibody that blocks PD-1 / PD-L1 interactions (MK3475) and a non-blocking anti-PD-1 antibody (135C H1cL1c (including SEQ ID NOs: 176 and 190) or 135C H1cL1b (SEQ ID NOs: 176 and 189). The effect of other combinations with)) is shown. As shown therein, the combination of the blocking antibody MK3475 and the non-blocking anti-PD-1 antibody (135C H1cL1c or 135C H1cL1b) has a statistically significant increase in proliferation compared to either antibody alone (shown). As shown above, p-value = 0.018 or p-value <0.0001). The data shown in FIG. 19 is the average of two experiments for each combination.

Example 9
Activation of Jurkat PD-1 reporter cells in combination with blocking anti-PD-1 antibody and non-blocking anti-PD-1 antibody In these experiments, the Jurkat PD-1NFAT reporter cell line was used with the TCR activator and PD-L1 protein. Stimulated with an expressed transiently transfected 293T cell line. In the absence of anti-PD-1 antibody, PD-L1 / PD-1 interactions suppress Jurkat cell stimulation, resulting in reduced activation of NFAT and lower levels of luciferase production. Addition of a blocking anti-PD-1 antibody (eg, MK3475) resulted in enhanced NFAT activation, more luciferase production, and an increase in the amount of chemiluminescence produced by the addition of the luciferase substrate. Under these extreme assay conditions with a large excess of PD-1 and PD-L1, non-blocking anti-PD-1 antibodies (represented by A35774 in FIG. 20A and 135C H1cL1c in FIG. 20B) are blocking anti-PD-. Has reduced activity compared to 1 antibody. However, the combination of blocking anti-PD-1 antibody and non-blocking anti-PD-1 antibody further enhances T cell activation to higher levels than achieved with MK3475 alone. This enhancement of NFAT activation with blocking and non-blocking anti-PD-1 antibodies is seen at both low and high concentrations of each test antibody. The data shown show a statistically significant increase in NFAT activation shown for any combination of blocking antibody MK3475 + non-blocking A35774 (FIG. 20A) or blocking antibody MK3475 + non-blocking antibody 135C H1cL1c (FIG. 20B). It is the average of 3 repeated experiments having.

Example 10
Internalization of cell surface PD-1 in anti-PD-1 antibody treatment Blocking anti-PD-1 antibody, non-blocking anti-PD-1 antibody, PBMC derived from chronically infected HIV donors with memory T cells with high basal levels of PD-1 Incubation with either PD-1 antibody or a combination of blocking anti-PD-1 antibody and non-blocking anti-PD-1 antibody identified effects on PD-1 internalization (FIG. 21). Monitor cell surface levels of PD-1 in memory CD4 ⁺ T cells (FIGS. 21a, c, and e) and CD8 ⁺ T cells (FIGS. 21b, d, and f) by flow cytometry over 48 to 72 hours. It was determined whether the anti-PD-1 antibody induces cell surface PD-1 internalization. A35795 (including SEQ ID NOs: 143 and 155) and A35774 (including SEQ ID NOs: 166 and 180) (FIGS. 21a, b); MK3475 and A35774 (including SEQ ID NOs: 166 and 180) (FIGS. 21c, d); and A35795. Three separate experiments are shown using a combination of antibodies (including SEQ ID NOs: 143 and 155) and 135C H1cL1c (including SEQ ID NOs: 176 and 190) (FIGS. 21, e, f). In all cases, blocking anti-PD-1 antibody (represented by A35795 or MK3475) and non-blocking anti-PD-1 antibody (A35774 (including SEQ ID NOs: 166 and 180) or 135C H1cL1c (SEQ ID NOs: 176 and 190)). In combination with) was observed to induce faster and / or more significant internalization of PD-1 receptors. In each experiment, PBMCs were incubated with human IgG4 isotype control antibody, which served as a reference for untreated T cells that maintained high levels of PD-1 throughout cell culture incubation. Mechanically, PD-1 internalization is anti-PD-, as reduced cell surface expression of PD-1 can limit the negative regulation of T cells through PD-1 / PD-L1 interactions. It can contribute to the antagonist functional activity of one antibody (either blocking and non-blocking antibody alone or in combination).

Example 11
IFN-γ production after anti-PD-1 antibody treatment PBMCs derived from chronically infected HIV donors with memory T cells with high basal levels of PD-1, blocking anti-PD-1 antibody, non-blocking anti-PD-1 antibody, Alternatively, it was incubated with any combination of blocking anti-PD-1 antibody and non-blocking anti-PD-1 antibody. The PBMC / antibody mixture was then laminated onto 293T cells transiently transfected to express the anti-CD3 TCR activator or co-express the anti-CD3 TCR activator and PD-L1. After 24-hour stimulation with TCR activator-expressing cells in the presence or absence of PD-L1, supernatants were collected and analyzed for IFN-γ production by ELISA. Similar levels of IFN-γ were detected in all antibody-treated samples when stimulated with anti-CD3 TCR activator-expressing cells (FIG. 22A). In contrast, when stimulated with cells co-expressing anti-CD3 TCR activator and PD-L1, lower levels of IFN-γ were detected in samples treated with IgG4 isotype control and samples treated with anti-PD-1 antibody. IFN-γ production was enhanced. The highest levels of cytokine production were observed in samples treated with both blocking anti-PD-1 antibody (MK3475) and non-blocking anti-PD-1 antibody (A35774) (* p <0.05).

In another experiment, PBMCs from chronically infected HIV donors with memory T cells with high basal levels of PD-1 were subjected to non-blocking anti-PD-1 antibody (A35774), blocking anti-PD-1 antibody (MK3475), or Incubated with any combination of blocking anti-PD-1 antibody and non-blocking anti-PD-1 antibody (MK3475 and A35774) (FIG. 22B). The PBMC / antibody mixture was then laminated onto 293T cells transiently transfected to co-express the anti-CD3 TCR activator and PD-L1 protein. A GolgiPlug ™ protein transport inhibitor was added and the cells were incubated for about 18 hours to accumulate cytokines within the cells. The proportion (%) of IFN-γ-producing cells was evaluated for each antibody treatment condition by flow cytometry (FIG. 22B). IFN-γ production was increased in PD-1 positive cells treated with anti-PD-1, and the combination of blocking antibody (MK3475) and non-blocking (A35774) antibody resulted in a further increase in cytokine production. (Data are shown for PD-1 ⁺ CD8 T cells).

Example 12
Crystal structure analysis Recombinantly expressed purified human PD-1 protein (receptor ectodomain residues 33-150 (“hPD1”)) is incubated with a Fab fragment of the humanized A35774 antibody and the complex is size exclusion chromatographed. Purified by. Protein crystals were prepared for the hPD-1 / Fab A35774 (including SEQ ID NOs: 166 and 180) complex, and the structure was elucidated by X-ray crystallography. The co-crystal structure of FIG. 23A includes heavy chains and heavy chains of A35774 (including SEQ ID NOs: 166 and 180, where CDRs are represented by SEQ ID NOs: 17, 40, 63, 86, 109, and 132) that interact with hPD-1. The light chain CDR loop is shown. The data show that the major residues that function as binding epitopes on PD-1 for A35774 are F37, P39, A40, L41, V43, L138, R139 and R143, and the _VL CDR loop for the _VH CDR loop. It is shown that P34, E136, S137 and R139 are included. These structural tests are consistent with epitope mapping performed using mouse 135C12 antibodies with the same heavy and light chain CDRs as A35774, as described in Examples 3 and 5. In their antibody binding experiments with PD-1 encoding amino acid substitutions, 135C12 antibodies and PD-1 mutants M4 (including substitutions S36A / P37A / L41A), M26 (with substitutions R139A / E141A); And a shift in binding between PD-1 mutant M31 (with substitution L141A / V143L) was observed. Other PD-1 mutations (in M17, M18 and M28) that resulted in a small shift in the binding affinity of 135C12 are in close proximity to the binding site of _A35774 Fab on PD-1 and these amino acids. Substitution can introduce local structural changes in PD-1 with reduced antibody binding affinity in these tests. Co-crystal structure analysis, epitope mapping, and antibody competitive binding tests are combined to distinguish antibody binding that overlaps the P2 patch region from antibodies that act via PD-1 / PD-L1 blockade. Provides confirmation that it can have activity. FIG. 23B provides additional evidence that the antibody represented by A35774 does not block the interaction between PD-1 and PD-L1. In FIG. 23B, a model was created between our hPD-1 / Fab _A35774 co-crystal structure and the co-crystal structure 4ZQK of PD-1 with PD-L1. This model shows non-overlapping binding of A35774F _ab and PD-L1 proteins to human PD-1 (FIG. 23B).

Example 13
In vivo Efficacy Test In to evaluate the therapeutic efficacy of non-blocking anti-PD-1 antibody 135C H1cL1c (including SEQ ID NOs: 176 and 190) in PD-1 HuGEMM mice compared to Keytruda (MK3475). Vivo efficacy studies were performed and each was administered bi-weekly at 10 mg / kg (FIG. 24). Additional therapeutic treatment involves co-administering 135C H1cL1c and Keytruda at 5 mg / kg each biweekly. HuGEMM mice were genetically engineered to express the human / mouse chimeric PD-1 protein in which the majority of the receptor ectodomain encodes the human PD-1 protein (residues 26-146 of the human PD-1 sequence). .. Prior to the in vivo test, flow cytometry confirmed that 135C H1cL1c and Keytruda anti-PD-1 antibody specifically bound to HuGEMM human / mouse chimeric PD-1 on the surface of stimulated T cells. .. In preparation for that study, a large number of PD-1HuGEMM mice were subcutaneously inoculated with MC38 cells from colorectal adenocarcinoma for tumor development. When the tumor reached a volume of about 95 mm ³ ± 50, a total of 40 mice were randomized into 4 groups with an average tumor size of 84 mm ³ and administered on day 0 of the study (vehicle, 135C H1cL1c,). Keytruda and 135C H1cL1c + Keytruda) were started. Therapeutic anti-PD-1 antibody was administered biweekly and tumor size and mouse body weight were measured twice a week. Tumor growth inhibition was monitored in contrast to untreated PBS buffer vehicle control mice and the effect was compared to Keytruda. Non-blocking anti-PD-1 antibody 135C H1cL1c induced a reduction in tumor growth at levels comparable to Keytruda. Co-administration of 135C H1cL1c with Keytruda also induced a reduction in cell proliferation that was at least as effective as Keytruda alone at 10 mg / kg. However, at all post-treatment time points during the study, mice treated with the combination of 135C H1cL1c and Keytruda showed improved response and a tendency for smaller mean tumor volume compared to Keytruda positive controls. Importantly, the combination of 135C H1cL1c and Keytruda was performed on day 4 (p-value = 0.0676, near statistical limit) and day 7 (using the Pearwise Wilcoxon test for statistical analysis). P-value = 0.0266), day 11 (p-value = 0.0067), day 14 (p-value = 0.0037), and day 17 (p-value = 0.0021) compared to vehicle control And showed a statistically significant decrease in tumor volume. In contrast, administration of Keytruda alone was performed on day 11 (p-value = 0.0205), day 14 (p-value = 0.0145), and day 17 (p-value = 0.0145) with the vehicle control. It only showed a statistically significant reduction in tumor volume in comparison. Administration of 135C H1cL1c alone was administered to the vehicle control on days 11 (p-value = 0.0266), 14 (p-value = 0.0062), and 17 (p-value = 0.0044) of the study. In comparison, it showed a statistically significant reduction in tumor volume (FIG. 24A). Interpretation of data on the average inhibition percentage (%) of MC38 tumor volume compared to vehicle controls also showed a strong increase in antitumor activity of the combination of 135C H1cL1c and Keytruda compared to Keytruda or 135C H1cL1c administered alone. The tendency is shown (FIG. 24B). In this profile, a combination of blocking (eg, cytokine or MK3475) anti-PD-1 antibody and non-blocking (135C H1cL1c) anti-PD-1 antibody is associated with T cell functional activity (eg, proliferation (eg, Examples 2 and 8)). It also supports in vitro evidence of causing an increase in cytokine production (Example 11)). Further findings were H1cL1c and Keytruda compared to Keytruda alone, despite the same total amount of antibody administered to each study group (Keytruda: 10 mg / kg, and 135C H1cL1c and Keytruda: 5 mg / kg each). The combination with is to begin to exert an antitumor effect at a fairly early stage (4th or 7th day of the study). Finally, in vivo tumor model data will be described for the percentage of mice that experienced complete remission of M38 tumors during antibody treatment (FIG. 24C). Consistent with the improvement in tumor inhibition (%) seen with the 135C H1cL1c plus Keytruda combination, this study group (135C H1cL1c plus Keytruda) doubled the increase in mice with complete remission of transplanted tumors. Shown (4 mice in a combination of 135C H1cL1c and Keytruda, as opposed to 2 mice in the group receiving either Keytruda or 135C H1cL1c individually / alone). Together, this in vivo data shows that binding to a PD-1 epitope that overlaps the P2 patch, which is non-blocking to PD-1 / PD-L1 interactions, activates a tumor-specific immune response. Is distinguished from and synonymous with anti-PD-1 antibodies that act through PD-1 / PD-L1 blockade.

Although the predetermined embodiments have been described for preferred embodiments, it should be appreciated that modifications and modifications can be conceived by those of skill in the art. Therefore, the appended claims are intended to cover all such equivalent modifications that fall within the claims.

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Claims (16)

A humanized antibody or fragment thereof that binds to human PD-1 and comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 176 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 190. The humanized antibody or fragment thereof according to claim 1, wherein the fragment is a _Fab , F (ab') ₂ , Fab', single-chain antibody or Fv fragment. The humanized antibody or fragment thereof according to claim 1 or 2, which is for inducing internalization of PD-1 from the surface of T cells. The humanized antibody or fragment thereof according to claim 3, wherein the T cell is a human T cell infected with a human immunodeficiency virus (HIV). The humanized antibody or fragment thereof according to claim 1 or 2, which is intended to relieve the functional exhaustion of antigen-specific CD8 ⁺ T cells and enhance their proliferation. The humanized antibody or fragment thereof according to claim 5, wherein the T cells are specific for the human immunodeficiency virus (HIV). The humanized antibody or fragment thereof according to claim 1 or 2, which is for treating cancer in a human patient. The humanized antibody or fragment thereof according to any one of claims 3 to 7, which is used in combination with the anti-PD-1 antibody pembrolizumab. The humanized antibody or fragment thereof according to claim 1 or 2, which is a binder having a second binding specificity to a different antigen. The derivative of the humanized antibody or fragment thereof according to claim 1 or 2, which comprises a detectable label or effector moiety fixedly attached to the humanized antibody or fragment thereof. A composition comprising the antibody or fragment thereof according to any one of claims 1 to 9, or the derivative according to claim 10, and at least one pharmaceutically acceptable carrier. A composition comprising the antibody or fragment thereof according to any one of claims 1 to 9, or the derivative according to claim 10, or the composition according to claim 11 further comprising the anti-PD-1 antibody pembrolizumab. An isolated polynucleotide encoding the humanized antibody or fragment thereof according to claim 1. An expression vector comprising one or more polynucleotides according to claim 13 . A host cell comprising the expression vector according to claim 14 . A host cell comprising one or more polynucleotides according to claim 13 .

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